Systemic delivery of murine SOD2 mRNA to experimental abdominal aortic aneurysm mitigates expansion and rupture.

Background: Oxidative stress is implicated in the pathogenesis and progression of abdominal aortic aneurysm (AAA). Antioxidant delivery as a therapeutic for AAA is of substantial interest although clinical translation of antioxidant therapy has met with significant challenges due to limitations in achieving sufficient antioxidant levels at the site of AAA. We posit that nanoparticle-based approaches hold promise to overcome challenges associated with systemic administration of antioxidants. Methods: We employed a peptide-based nanoplatform to overexpress a key modulator of oxidative stress, superoxide dismutase 2 (SOD2). The efficacy of systemic delivery of SOD2 mRNA as a nanotherapeutic agent was studied in two different murine AAA models. Unbiased mass spectrometry-enabled proteomics and high-dimensional bioinformatics were used to examine pathways modulated by SOD2 overexpression. Results: The murine SOD2 mRNA sequence was mixed with p5RHH, an amphipathic peptide capable of delivering nucleic acids in vivo to form self-assembled nanoparticles of ∼55 nm in diameter. We further demonstrated that the nanoparticle was stable and functional up to four weeks following self-assembly when coated with hyaluronic acid. Delivery of SOD2 mRNA mitigated the expansion of small AAA and largely prevented rupture. Mitigation of AAA was accompanied by enhanced SOD2 protein expression in aortic wall tissue. Concomitant suppression of nitric oxide, inducible nitric oxide synthase expression, and cell death was observed. Proteomic profiling of AAA tissues suggests that SOD2 overexpression augments levels of microRNAs that regulate vascular inflammation and cell apoptosis, inhibits platelet activation/aggregation, and downregulates mitogen-activated protein kinase signaling. Gene set enrichment analysis shows that SOD2 mRNA delivery is associated with activation of oxidative phosphorylation, lipid metabolism, respiratory electron transportation, and tricarboxylic acid cycle pathways. Conclusions: These results confirm that SOD2 is key modulator of oxidative stress in AAA. This nanotherapeutic mRNA delivery approach may find translational application in the medical management of small AAA and the prevention of AAA rupture.

Inherited C-terminal TREX1 variants disrupt homology-directed repair to cause senescence and DNA damage phenotypes in Drosophila, mice, and humans.

Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, we reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, we reasoned that nuclear TREX1 would cause DNA damage. Here, we show that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, we observe early-onset breast cancer, similar to patients with BRCA1/2 variants. Our results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.

Transcriptomic profiling of thymic dysregulation and viral tropism after neonatal roseolovirus infection.

Introduction: Herpesviruses, including the roseoloviruses, have been linked to autoimmune disease. The ubiquitous and chronic nature of these infections have made it difficult to establish a causal relationship between acute infection and subsequent development of autoimmunity. We have shown that murine roseolovirus (MRV), which is highly related to human roseoloviruses, induces thymic atrophy and disruption of central tolerance after neonatal infection. Moreover, neonatal MRV infection results in development of autoimmunity in adult mice, long after resolution of acute infection. This suggests that MRV induces durable immune dysregulation. Methods: In the current studies, we utilized single-cell RNA sequencing (scRNAseq) to study the tropism of MRV in the thymus and determine cellular processes in the thymus that were disrupted by neonatal MRV infection. We then utilized tropism data to establish a cell culture system. Results: Herein, we describe how MRV alters the thymic transcriptome during acute neonatal infection. We found that MRV infection resulted in major shifts in inflammatory, differentiation and cell cycle pathways in the infected thymus. We also observed shifts in the relative number of specific cell populations. Moreover, utilizing expression of late viral transcripts as a proxy of viral replication, we identified the cellular tropism of MRV in the thymus. This approach demonstrated that double negative, double positive, and CD4 single positive thymocytes, as well as medullary thymic epithelial cells were infected by MRV in vivo. Finally, by applying pseudotime analysis to viral transcripts, which we refer to as "pseudokinetics," we identified viral gene transcription patterns associated with specific cell types and infection status. We utilized this information to establish the first cell culture systems susceptible to MRV infection in vitro. Conclusion: Our research provides the first complete picture of roseolovirus tropism in the thymus after neonatal infection. Additionally, we identified major transcriptomic alterations in cell populations in the thymus during acute neonatal MRV infection. These studies offer important insight into the early events that occur after neonatal MRV infection that disrupt central tolerance and promote autoimmune disease.

Uropathogenic Escherichia coli infection-induced epithelial trained immunity impacts urinary tract disease outcome.

Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding-responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.

Rare missense variants in the SH3 domain of PSTPIP1 are associated with hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic, debilitating skin disease for which few treatment options are available. While most HS is sporadic, some rare kindred show a high-penetrance, autosomal-dominant inheritance. We wanted to identify rare variants that could contribute to HS risk in sporadic cases using candidate gene sequencing. We ultimately identified 21 genes for our capture panel. We included genes of the γ-secretase complex (n = 6) because rare variants in these genes sometimes cause familial HS. We added Notch receptor and ligand genes (n = 13) because γ-secretase is critical for processing Notch receptor signaling. Clinically, some people with PAPA (pyogenic arthritis, pyoderma gangrenosum, and acne) syndrome, a rare inflammatory disease, have concurrent HS. Rare variants in PSTPIP1 are known to cause PAPA syndrome, so we included PSTPIP1 and PSTPIP2 in the capture panel. We screened 117 individuals with HS for rare variations and calculated the expected burden using Genome Aggregation Database (gnomAD) allele frequencies. We discovered two pathogenic loss-of-function variants in NCSTN. This class of NCSTN variant can cause familial HS. There was no increased burden of rare variations in any γ-secretase complex gene. We did find that individuals with HS had a significantly increased number of rare missense variants in the SH3 domain of PSTPIP1. This finding, therefore, implicates PSTPIP1 variation in sporadic HS and further supports dysregulated immunity in HS. Our data also suggests that population-scale HS genetic research will yield valuable insights into disease pathology.

Alterations of the Primary Cilia Gene SPAG17 and SOX9 Locus Noncoding RNAs Identified by RNA-Sequencing Analysis in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by immune activation, vasculopathy, and unresolving fibrosis in the skin, lungs, and other organs. We performed RNA-sequencing analysis on skin biopsy samples and peripheral blood mononuclear cells (PBMCs) from SSc patients and unaffected controls to better understand the pathogenesis of SSc. METHODS: We analyzed these data 1) to test for case/control differences and 2) to identify genes whose expression levels correlate with SSc severity as measured by local skin score, modified Rodnan skin thickness score (MRSS), forced vital capacity (FVC), or diffusing capacity for carbon monoxide (DLco). RESULTS: We found that PBMCs from SSc patients showed a strong type I interferon signature. This signal was found to be replicated in the skin, with additional signals for increased extracellular matrix (ECM) genes, classical complement pathway activation, and the presence of B cells. Notably, we observed a marked decrease in the expression of SPAG17, a cilia component, in SSc skin. We identified genes that correlated with the MRSS, DLco, and FVC in SSc PBMCs and skin using weighted gene coexpression network analysis. These genes were largely distinct from the case/control differentially expressed genes. In PBMCs, type I interferon signatures negatively correlated with the DLco. In SSc skin, ECM gene expression positively correlated with the MRSS. Network analysis of SSc skin genes that correlated with clinical features identified the noncoding RNAs SOX9-AS1 and ROCR, both near the SOX9 locus, as highly connected, "hub-like" genes in the network. CONCLUSION: These results identify noncoding RNAs and SPAG17 as novel factors potentially implicated in the pathogenesis of SSc.

Reduced SPAG17 Expression in Systemic Sclerosis Triggers Myofibroblast Transition and Drives Fibrosis.

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-ß pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.

Inhibition of miRNA associated with a disease-specific signature and secreted via extracellular vesicles of systemic lupus erythematosus patients suppresses target organ inflammation in a humanized mouse model.

Introduction: Distinct, disease-associated intracellular miRNA (miR) expression profiles have been observed in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients. Additionally, we have identified novel estrogenic responses in PBMCs from SLE patients and demonstrated that estrogen upregulates toll-like receptor (TLR)7 and TLR8 expression. TLR7 and TLR8 bind viral-derived single-stranded RNA to stimulate innate inflammatory responses, but recent studies have shown that miR-21, mir-29a, and miR-29b can also bind and activate these receptors when packaged and secreted in extracellular vesicles (EVs). The objective of this study was to evaluate the association of EV-encapsulated small RNA species in SLE and examine the therapeutic approach of miR inhibition in humanized mice. Methods: Plasma-derived EVs were isolated from SLE patients and quantified. RNA was then isolated and bulk RNA-sequencing reads were analyzed. Also, PBMCs from active SLE patients were injected into immunodeficient mice to produce chimeras. Prior to transfer, the PBMCs were incubated with liposomal EVs containing locked nucleic acid (LNA) antagonists to miR-21, mir-29a, and miR-29b. After three weeks, blood was collected for both immunophenotyping and cytokine analysis; tissue was harvested for histopathological examination. Results: EVs were significantly increased in the plasma of SLE patients and differentially expressed EV-derived small RNA profiles were detected compared to healthy controls, including miR-21, mir-29a, and miR-29b. LNA antagonists significantly reduced proinflammatory cytokines and histopathological infiltrates in the small intestine, liver, and kidney, as demonstrated by H&E-stained tissue sections and immunohistochemistry measuring human CD3. Discussion: These data demonstrate distinct EV-derived small RNA signatures representing SLE-associated biomarkers. Moreover, targeting upregulated EV-encapsulated miR signaling by antagonizing miRs that may bind to TLR7 and TLR8 reveals a novel therapeutic opportunity to suppress autoimmune-mediated inflammation and pathogenesis in SLE.

The CSF in neurosarcoidosis contains consistent clonal expansion of CD8 T cells, but not CD4 T cells.

The tissue-specific drivers of neurosarcoidosis remain poorly defined. To identify cerebrospinal fluid (CSF) specific, antigen-driven T and B cell responses, we performed single-cell RNA sequencing of CSF and blood cells from neurosarcoid participants coupled to T and B cell receptor sequencing. In contrast to pulmonary sarcoidosis, which is driven by CD4 T cells, we found CD8 T cell clonal expansion enriched in the neurosarcoid CSF. These CSF-enriched CD8 T cells were composed of two subsets with differential expression of EBI2, CXCR3, and CXCR4. Lastly, our data suggest that IFNγ signaling may distinguish neurosarcoidosis from other neurological disorders.

Transcriptomes of peripheral blood mononuclear cells from juvenile dermatomyositis patients show elevated inflammation even when clinically inactive.

In juvenile dermatomyositis (JDM), the most common pediatric inflammatory myopathy, weakness is accompanied by a characteristic rash that often becomes chronic and is associated with vascular damage. We hoped to understand the molecular underpinnings of JDM, particularly when untreated, which would facilitate the identification of novel mechanisms and clinical targets that might disrupt disease progression. We studied the RNA-Seq data from untreated JDM peripheral blood mononuclear cells (PBMCs; n = 11), PBMCs from a subset of the same patients when clinically inactive (n = 8/11), and separate samples of untreated JDM skin and muscle (n = 4 each). All JDM samples were compared to non-inflammatory control tissues. The untreated JDM PBMCs showed a strong signature for type1 interferon response, along with IL-1, IL-10, and NF-κB. Surprisingly, PBMCs from clinically inactive JDM individuals had persistent immune activation that was enriched for IL-1 signaling. JDM skin and muscle both showed evidence for type 1 interferon activation and genes related to antigen presentation and decreased expression of cellular respiration genes. Additionally, we found that PBMC gene expression correlates with disease activity scores (DAS; skin, muscle, and total domains) and with nailfold capillary end row loop number (an indicator of microvascular damage). This included otoferlin, which was significantly increased in untreated JDM PBMCs and correlated with all 3 DAS domains. Overall, these data demonstrate that PBMC transcriptomes are informative of molecular disruptions in JDM and provide transcriptional evidence of chronic inflammation despite clinical quiescence.

STING Gain-of-Function Disrupts Lymph Node Organogenesis and Innate Lymphoid Cell Development in Mice.

STING gain-of-function causes autoimmunity and immunodeficiency in mice and STING-associated vasculopathy with onset in infancy (SAVI) in humans. Here, we report that STING gain-of-function in mice prevents development of lymph nodes and Peyer's patches. We show that the absence of secondary lymphoid organs is associated with diminished numbers of innate lymphoid cells (ILCs), including lymphoid tissue inducer (LTi) cells. Although wild-type (WT) α4ß7+ progenitors differentiate efficiently into LTi cells, STING gain-of-function progenitors do not. Furthermore, STING gain-of-function impairs development of all types of ILCs. Patients with STING gain-of-function mutations have fewer ILCs, although they still have lymph nodes. In mice, expression of the STING mutant in RORγT-positive lineages prevents development of lymph nodes and reduces numbers of LTi cells. RORγT lineage-specific expression of STING gain-of-function also causes lung disease. Since RORγT is expressed exclusively in LTi cells during fetal development, our findings suggest that STING gain-of-function prevents lymph node organogenesis by reducing LTi cell numbers in mice.

Dynamic immunoglobulin responses to gut bacteria during inflammatory bowel disease.

Aberrant immune responses against gut microbiota are thought to be key drivers of inflammatory bowel disease (IBD) pathogenesis. However, the extent and targets of immunoglobulin (Ig) A versus IgG responses to gut bacteria in IBD and its association with IBD severity is not well understood. Here, we address this by analyzing fecal samples from Crohn's disease (CD), ulcerative colitis (UC), and Non-IBD patients by flow cytometry for the frequency of bacteria that were endogenously bound with IgA and/or IgG. Assessment of IBD patients from two geographically distinct cohorts revealed increased percentages of IgA- and IgG-bound fecal bacteria compared to non-IBD controls. Notably, the two major subsets of IBD showed distinct patterns of Ig-bound bacteria, with CD activity associated with increases in both IgA and IgG-bound bacteria, whereas UC activity correlated only with increases in IgG-bound bacteria. Analysis of the flow sorted Ig-bound bacterial repertoire by 16S rDNA sequencing revealed taxa that were Ig-bound specifically in IBD. Notably, this included bacteria that are also thought to reside in the oral pharynx, including Gemella, Peptostreptococcus, and Streptococcus species. These data show that the pattern of IgA and IgG binding to fecal bacteria is distinct in UC and CD. In addition, the frequency of Ig-bound fecal bacteria may have potential as a non-invasive biomarker for disease activity. Finally, our results support the hypothesis that immune responses to oral pharyngeal bacteria may play an important role in the pathogenesis of IBD.

A catalog of CasX genome editing sites in common model organisms.

DpbCasX, also called Cas12e, is an RNA-guided DNA endonuclease isolated from Deltaproteobacteria. In this paper I characterized the CasX-compatible genome editing sites in the reference genomes of yeast (Saccharomyces cerevisiae), flatworms (Caenorhabditis elegans), flies (Drosophila melanogaster), zebrafish (Danio rerio), mouse (Mus musculus), rats (Rattus norvegicus), and humans (Homo sapiens). Across those genomes there were > 27,000 CasX sites per megabase on average. More than 90% of genes in each genome had at least one unique site overlapping an exon, with median unique sites per gene of 6-45. I also annotated sites in the GRCm38 reference and 15 additional mouse strain genomes. The presence of specific guide sequences varied amongst the strains, with CAST/EiJ and PWK/PhJ showing the greatest divergence from the reference strain. The high density of CasX sites and number of exon overlapping sites suggests that CasX has the potential to be used as a common genome editor.

Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot.

Nicastrin (NCSTN) is a transmembrane glycoprotein that is part of the gamma-secretase complex. Gamma-secretase is a protease complex that cleaves type-I single-pass transmembrane proteins. There are many potential substrates for this complex, including NOTCH receptors and amyloid precursor proteins (APP). There are a number of commercial antibodies to nicastrin, but they do not agree on expected peptide size. We confirmed the specificity of a C-terminal binding rabbit anti-human antibody from Sigma-Aldrich (#N1660) using wildtype HEK293 cells and HEK293 cells deleted for nicastrin. The wildtype cells showed a prominent band at approximately 110 kDa. We confirmed this larger than expected sized was due to glycosylation by treating the lysate with peptide-N-glycosidase F (PNGase F), which reduced the band to less than 75 kDa. These data suggest that this polyclonal is specific for nicastrin and can detect endogenous levels of protein.

Motif scraper: a cross-platform, open-source tool for identifying degenerate nucleotide motif matches in FASTA files.

Summary: Many genomic features are defined not by exact sequence matches, but by degenerate nucleotide motifs that represent multiple compatible matches. While there are databases cataloging genomic features, such as the location of transcription factor motifs, for commonly used model species, identifying the locations of novel motifs, known motifs in non-model genomes, or known motifs in personal whole-genomes is difficult. I designed motif scraper to overcome this limitation, allowing for efficient, multiprocessor motif searches in any FASTA file. Availability and implementation: The motif scraper package (MIT license) is available via PyPI, and the Python source is available on GitHub at https://github.com/RobersonLab/motif_scraper.

Protective Low-Frequency Variants for Preeclampsia in the Fms Related Tyrosine Kinase 1 Gene in the Finnish Population.

Preeclampsia is a common pregnancy-specific vascular disorder characterized by new-onset hypertension and proteinuria during the second half of pregnancy. Predisposition to preeclampsia is in part heritable. It is associated with an increased risk of cardiovascular disease later in life. We have sequenced 124 candidate genes implicated in preeclampsia to pinpoint genetic variants contributing to predisposition to or protection from preeclampsia. First, targeted exomic sequencing was performed in 500 preeclamptic women and 190 controls from the FINNPEC cohort (Finnish Genetics of Preeclampsia Consortium). Then 122 women with a history of preeclampsia and 1905 parous women with no such history from the National FINRISK Study (a large Finnish population survey on risk factors of chronic, noncommunicable diseases) were included in the analyses. We tested 146 rare and low-frequency variants and found an excess (observed 13 versus expected 7.3) nominally associated with preeclampsia (P<0.05). The most significantly associated sequence variants were protective variants rs35832528 (E982A; P=2.49E-4; odds ratio=0.387) and rs141440705 (R54S; P=0.003; odds ratio=0.442) in Fms related tyrosine kinase 1. These variants are enriched in the Finnish population with minor allele frequencies 0.026 and 0.017, respectively. They may also be associated with a lower risk of heart failure in 11 257 FINRISK women. This study provides the first evidence of maternal protective genetic variants in preeclampsia.

A mucosal imprint left by prior Escherichia coli bladder infection sensitizes to recurrent disease.

Recurrent bacterial infections are a significant burden worldwide, and prior history of infection is often a significant risk factor for developing new infections. For urinary tract infection (UTI), a history of two or more episodes is an independent risk factor for acute infection. However, mechanistic knowledge of UTI pathogenesis has come almost exclusively from studies in naive mice. Here we show that, in mice, an initial Escherichia coli UTI, whether chronic or self-limiting, leaves a long-lasting molecular imprint on the bladder tissue that alters the pathophysiology of subsequent infections, affecting host susceptibility and disease outcome. In bladders of previously infected versus non-infected, antibiotic-treated mice, we found (1) an altered transcriptome and defects in cell maturation, (2) a remodelled epithelium that confers resistance to intracellular bacterial colonization, and (3) changes to cyclooxygenase-2-dependent inflammation. Furthermore, in mice with a history of chronic UTI, cyclooxygenase-2-dependent inflammation allowed a variety of clinical E. coli isolates to circumvent intracellular colonization resistance and cause severe recurrent UTI, which could be prevented by cyclooxygenase-2 inhibition or vaccination. This work provides mechanistic insight into how a history of infection can impact the risk for developing recurrent infection and has implications for the development of therapeutics for recurrent UTI.

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.