Erin K. Wagner

Rare genetic variants in the CFI gene are associated with advanced age-related macular degeneration and commonly result in reduced serum factor I levels

To assess a potential diagnostic and therapeutic biomarker for age-related macular degeneration (AMD), we sequenced the complement factor I gene (CFI) in 2266 individuals with AMD and 1400 without, identifying 231 individuals with rare genetic variants. We evaluated the functional impact by measuring circulating serum factor I (FI) protein levels in individuals with and without rare CFI variants. The burden of very rare (frequency <1/1000) variants in CFI was strongly associated with disease (P = 1.1 × 10(-8)). In addition, we examined eight coding variants with counts ≥5 and saw evidence for association with AMD in three variants. Individuals with advanced AMD carrying a rare CFI variant had lower mean FI compared with non-AMD subjects carrying a variant (P < 0.001). Further new evidence that FI levels drive AMD risk comes from analyses showing individuals with a CFI rare variant and low FI were more likely to have advanced AMD (P = 5.6 × 10(-5)). Controlling for covariates, low FI increased the risk of advanced AMD among those with a variant compared with individuals without advanced AMD with a rare CFI variant (OR 13.6, P = 1.6 × 10(-4)), and also compared with control individuals without a rare CFI variant (OR 19.0, P = 1.1 × 10(-5)). Thus, low FI levels are strongly associated with rare CFI variants and AMD. Enhancing FI activity may be therapeutic and measuring FI provides a screening tool for identifying patients who are most likely to benefit from complement inhibitory therapy.

Rare Variants in the Functional Domains of Complement Factor H Are Associated With Age-Related Macular Degeneration

PURPOSE Age-related macular degeneration (AMD) has a substantial genetic risk component, as evidenced by the risk from common genetic variants uncovered in the first genome-wide association studies. More recently, it has become apparent that rare genetic variants also play an independent role in AMD risk. We sought to determine if rare variants in complement factor H (CFH) played a role in AMD risk. METHODS We had previously collected DNA from a large population of patients with advanced age-related macular degeneration (A-AMD) and controls for targeted deep sequencing of candidate AMD risk genes. In this analysis, we tested for an increased burden of rare variants in CFH in 1665 cases and 752 controls from this cohort. RESULTS We identified 65 missense, nonsense, or splice-site mutations with a minor allele frequency ≤ 1%. Rare variants with minor allele frequency ≤ 1% (odds ratio [OR] = 1.5, P = 4.4 × 10⁻²), 0.5% (OR = 1.6, P = 2.6 × 10⁻²), and all singletons (OR = 2.3, P = 3.3 × 10⁻²) were enriched in A-AMD cases. Moreover, we observed loss-of-function rare variants (nonsense, splice-site, and loss of a conserved cysteine) in 10 cases and serum levels of FH were decreased in all 5 with an available sample (haploinsufficiency). Further, rare variants in the major functional domains of CFH were increased in cases (OR = 3.2; P = 1.4 × 10⁻³) and the magnitude of the effect correlated with the disruptive nature of the variant, location in an active site, and inversely with minor allele frequency. CONCLUSIONS In this large A-AMD cohort, rare variants in the CFH gene were enriched and tended to be located in functional sites or led to low serum levels. These data, combined with those indicating a similar, but even more striking, increase in rare variants found in CFI, strongly implicate complement activation in A-AMD etiopathogenesis as CFH and CFI interact to inhibit the alternative pathway.

Tissue-specific Co-expression of Long Non-coding and Coding RNAs Associated with Breast Cancer.

Inference of the biological roles of lncRNAs in breast cancer development remains a challenge. Here, we analyzed RNA-seq data in tumor and normal breast tissue samples from 18 breast cancer patients and 18 healthy controls and constructed a functional lncRNA-mRNA co-expression network. We revealed two distinctive co-expression patterns associated with breast cancer, reflecting different underlying regulatory mechanisms: (1) 516 pairs of lncRNA-mRNAs have differential co-expression pattern, in which the correlation between lncRNA and mRNA expression differs in tumor and normal breast tissue; (2) 291 pairs have dose-response co-expression pattern, in which the correlation is similar, but the expression level of lncRNA or mRNA differs in the two tissue types. We further validated our findings in TCGA dataset and annotated lncRNAs using TANRIC. One novel lncRNA, AC145110.1 on 8p12, was found differentially co-expressed with 127 mRNAs (including TOX4 and MAEL) in tumor and normal breast tissue and also highly correlated with breast cancer clinical outcomes. Functional enrichment and pathway analyses identified distinct biological functions for different patterns of co-expression regulations. Our data suggested that lncRNAs might be involved in breast tumorigenesis through the modulation of gene expression in multiple pathologic pathways.

Long intergenic non-coding RNA expression signature in human breast cancer.

Breast cancer is a complex disease, characterized by gene deregulation. There is less systematic investigation of the capacity of long intergenic non-coding RNAs (lincRNAs) as biomarkers associated with breast cancer pathogenesis or several clinicopathological variables including receptor status and patient survival. We designed a two-stage study, including 1,000 breast tumor RNA-seq data from The Cancer Genome Atlas (TCGA) as the discovery stage, and RNA-seq data of matched tumor and adjacent normal tissue from 50 breast cancer patients as well as 23 normal breast tissue from healthy women as the replication stage. We identified 83 lincRNAs showing the significant expression changes in breast tumors with a false discovery rate (FDR) < 1% in the discovery dataset. Thirty-seven out of the 83 were validated in the replication dataset. Integrative genomic analyses suggested that the aberrant expression of these 37 lincRNAs was probably related with the expression alteration of several transcription factors (TFs). We observed a differential co-expression pattern between lincRNAs and their neighboring genes. We found that the expression levels of one lincRNA (RP5-1198O20 with Ensembl ID ENSG00000230615) were associated with breast cancer survival with P < 0.05. Our study identifies a set of aberrantly expressed lincRNAs in breast cancer.

Abstract 3268: Differences in expression of lncRNAs in breast tumor, adjacent normal-appearing breast tissue, and normal breast tissue from healthy donors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Long noncoding RNAs (lncRNAs) are an emerging class of key regulatory RNAs that are greater than 200nt and do not code for protein. They appear to be critical for various biological processes including gene regulation. Increasing evidence has suggested that aberrantly expressed lncRNAs play a key role in the initiation and progression of breast cancer. In most studies of breast cancer, breast tumor tissue was compared to adjacent normal-appearing tissue to identify differentially expressed lncRNAs. However, expression of lncRNAs in normal breast tissue from healthy donors has rarely been studied, and the differences in expression of lncRNAs in breast tumor and normal breast tissue from healthy donors remain largely unknown. Using RNA-sequencing data generated from freshly-frozen breast tissue samples, we examined the expression of lncRNAs in 119 breast tumor, 47 adjacent normal-appearing breast tissue samples, and 23 normal breast tissue samples from healthy donors. Bowtie and RESM algorithms were used to align raw reads and quantify transcript abundance. Differential expression analyses were performed using edgeR software. We assessed the expression of a total of 7,844 mapped lncRNAs using normalized transcript counts. Unsupervised Principal Components Analysis (PCA) demonstrated that the lncRNA expression profile of adjacent normal-appearing tissue was different compared to normal breast tissue from healthy donors, and partially overlapped with the tumor. At various fold change thresholds, more differentially expressed lncRNAs can be detected when the tumor tissue is compared to normal tissue than to adjacent non-tumorous tissue. With a false discovery rate (FDR) < 0.05 and a fold change (FC) of more than ±2, we identified 107 differentially expressed lncRNAs for the tumor vs. normal breast tissue, 53 lncRNAs for tumor vs. adjacent normal-appearing tissue, and 20 lncRNAs for adjacent normal-appearing vs. normal breast tissue. In our analyses of tumor vs. adjacent normal-appearing tissue and tumor vs. normal tissue from healthy donors, we identified differentially expressed lncRNAs including HOTAIR, GAS5 and ANRIL/CDKN2B-AS1 that were previously implicated in breast cancer, with considerably higher fold changes in latter comparison. When using normal breast tissue from healthy donors as the baseline, we identified novel putative breast cancer-associated lncRNAs including EMX2OS, TERC, HOTAIRM1, and WT1-AS that are aberrantly expressed in breast tumor. These lncRNAs were not identifiable in a direct comparison of tumor and adjacent normal-appearing tissue. Our results suggest that adjacent normal-appearing tissue undergoes changes in lncRNAs expression similar to those observed in tumor tissue to some extent, and a number of new and potentially important lncRNAs can only be detected using normal tissue from healthy donors as an optimal baseline tissue. Citation Format: Erin Wagner, Yunlong Liu, Bryan Schneider, Anna Maria Storniolo, Jiali Han, Chunyan He. Differences in expression of lncRNAs in breast tumor, adjacent normal-appearing breast tissue, and normal breast tissue from healthy donors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3268. doi:10.1158/1538-7445.AM2014-3268

Protective coding variants in CFH and PELI3 and a variant near CTRB1 are associated with age-related macular degeneration†.

Although numerous common age-related macular degeneration (AMD) alleles have been discovered using genome-wide association studies, substantial disease heritability remains unexplained. We sought to identify additional common and rare variants associated with advanced AMD. A total of 4,332 cases and 25,268 controls of European ancestry from three different populations were genotyped using the Illumina Infinium HumanExome BeadChip. We performed meta-analyses to identify associations with common variants, and single variant and gene-based burden tests to identify rare variants. Two protective, low-frequency, non-synonymous variants were significantly associated with a decrease in AMD risk: A307V in PELI3 (odds ratio [OR] = 0.14, P = 4.3 × 10-10) and N1050Y in CFH (OR = 0.76, P = 6.2 × 10-12). The new variants have a large effect size, similar to some rare mutations we reported previously in a targeted sequencing study, which remain significant in this analysis: CFH R1210C (OR = 18.82, P = 3.5 × 10-07), C3 K155Q (OR = 3.27, P = 1.5 × 10-10) and C9 P167S (OR = 2.04, P = 2.8 × 10-07). We also identified a strong protective signal for a common variant (rs8056814) near CTRB1 associated with a decrease in AMD risk (logistic regression: OR = 0.71, P = 1.8 × 10-07). Suggestive protective loci were identified in the COL4A3 and APOH genes. Our results support the involvement of common and low-frequency protective variants in this vision-threatening condition. This study expands the roles of the innate immune pathway as well as the extracellular matrix and high-density lipoprotein pathways in the aetiology of AMD.

Novel protective associations with age-related macular degeneration: A common variant near CTRB1 and a rare variant in PELI3

Although >20 common frequency age-related macular degeneration (AMD) alleles have been discovered with genome-wide association studies, substantial disease heritability remains unexplained. In this study we sought to identify additional variants, both common and rare, that have an association with advanced AMD. We genotyped 4,332 cases and 4,642 controls of European ancestry from three different populations using the Illumina Infinium HumanExome BeadChip. We performed meta-analyses to identify associations with common variants and performed single variant and gene-based burden tests to identify associations with rare variants. We identified a novel rare (minor allele frequency < 1%) non-synonymous variant; A307V in the PELI3 gene (odds ratio [OR]=0.27, P=5.6x10-07). Additionally we identified an enrichment of protective alleles in PELI3 using a burden test (OR=0.28). The new rare variant has a large effect size, similar to rare mutations we reported previously in a targeted sequencing study, which remain significant in this analysis: CFH R1210C (OR=18.82, P=3.5x10-07), CFH N1050Y (OR=0.40, P=8.0x10-13), C3 K155Q (OR=3.27, P=1.5x10-10), and C9 P167S (OR=2.04, P=2.8x10-07). We also identified a novel common variant (rs8056814) near CTRB1 significantly associated with a decrease in AMD risk (odds ratio=0.71, P=7.7x10-07). This study supports the involvement of both common and rare protective variants in AMD. It also may expand the role of the high-density lipoprotein pathway and branches of the innate immune pathway, outside that of the complement system, in the etiology of AMD.Although >20 common frequency age-related macular degeneration (AMD) alleles have been discovered with genome-wide association studies, substantial disease heritability remains unexplained. In this study we sought to identify additional variants, both common and rare, that have an association with advanced AMD. We genotyped 4,332 cases and 25,268 controls of European ancestry from three different populations using the Illumina Infinium HumanExome BeadChip. We performed meta-analyses to identify associations with common variants and performed single variant and gene-based burden tests to identify associations with rare variants. Two protective, low frequency, non-synonymous variants A307V in PELI3 (odds ratio [OR]=0.14, P=4.3×10−10) and N1050Y in CFH (OR=0.76, Pconditional=1.6×10−11) were significantly associated with a decrease in risk of AMD. Additionally, we identified an enrichment of protective alleles in PELI3 using a burden test (OR=0.14). The new variants have a large effect size, similar to rare mutations we reported previously in a targeted sequencing study, which remain significant in this analysis: CFH R1210C (OR=18.82, P=3.5×10−07), C3 K155Q (OR=3.27, P=1.5×10−10), and C9 P167S (OR=2.04, P=2.8×10−07). We also identified a strong protective signal for a common variant (rs8056814) near CTRB1 associated with a decrease in AMD risk (logistic regression: OR = 0.71, P = 1.8x10−07; Firth corrected OR = 0.64, P = 9.6x10−11). This study supports the involvement of both common and low frequency protective variants in AMD. It also may expand the role of the high-density lipoprotein pathway and branches of the innate immune pathway, outside that of the complement system, in the etiology of AMD.