Purpose: To develop a genotype assay to assess associations with common and rare age-related macular degeneration (AMD) risk variants, to calculate an overall genetic risk score (GRS), and to identify potential misdiagnoses with inherited macular dystrophies that mimic AMD.Design: Case-control study.Participants: Individuals (n ¼ 4740) from 5 European cohorts. Methods: We designed single-molecule molecular inversion probes for target selection and used next generation sequencing to sequence 87 single nucleotide polymorphisms (SNPs), coding and splice-site regions of 10 AMD-(related) genes (ARMS2, C3, C9, CD46, CFB, CFH, CFI, HTRA1, TIMP3, and SLC16A8), and 3 genes that cause inherited macular dystrophies (ABCA4, CTNNA1, and PRPH2). Genetic risk scores for common AMD risk variants were calculated based on effect size and genotype of 52 AMD-associated variants. Frequency of rare variants was compared between late AMD patients and control individuals with logistic regression analysis.Main Outcome Measures: Genetic risk score, association of genetic variants with AMD, and genotypeephenotype correlations.Results: We observed high concordance rates between our platform and other genotyping platforms for the 69 successfully genotyped SNPs (>96%) and for the rare variants (>99%). We observed a higher GRS for patients with late AMD compared with patients with early/intermediate AMD (P < 0.001) and individuals without AMD (P < 0.001). A higher proportion of pathogenic variants in the CFH (odds ratio [OR] ¼ 2.88; P ¼ 0.006), CFI (OR ¼ 4.45; P ¼ 0.005), and C3 (OR ¼ 6.56; P ¼ 0.0003) genes was observed in late AMD patients compared with control individuals. In 9 patients, we identified pathogenic variants in the PRPH2, ABCA4, and CTNNA1 genes, which allowed reclassification of these patients as having inherited macular dystrophy.Conclusions: This study reports a genotype assay for common and rare AMD genetic variants, which can identify individuals at intermediate to high genetic risk of late AMD and enables differential diagnosis of AMDmimicking dystrophies. Our study supports sequencing of CFH, CFI, and C3 genes because they harbor rare high-risk variants. Carriers of these variants could be amendable for new treatments for AMD that currently are under development. Ophthalmology 2020;-:1e14

Abstract Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G&gt;A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD, however its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118W and p.P167S) C9 variants.Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared to non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and ARPE-19 cells by carriers' sera. Our data suggest that the analysed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.

No abstract

Objective To investigate the reasons for cone biopsies reported as not containing intraepithelial or invasive malignancy and thereby find ways to decrease their incidence.Design One hundred cone biopsies reported as negative were identified out of a total of 436 consecutive cone biopsies. The patients' cytology, colposcopy and histology reports and cytology and histology slides were reviewed. Further opinions in cases of doubt were obtained in cytology and histology. In cone biopsies still considered negative after reviews, deeper levels were cut, exhausting all paraffin blocks. Follow up cytology, colposcopy and histology were reviewed.Setting Gynaecological oncology unit in a university teaching hospital.Results After re-evaluation the final diagnoses of cone biopsies initially reported as negative were positive (n = 21), unsatisfactory (n = 27) and true negative (n = SI), with one case excluded because of insufficient material for review. The positive cases were diagnosed on review (n = 11) or extra levels (n = 10). The unsatisfactory cases were all due to denudation. The 5 1 true negative cases were divided into those which never had had histologic confirmation by punch biopsy or endocervical curettage (n = 47) and those with a previously confirmed histological abnormality (n = 4). Conclusions The number of negative cone biopsies can be reduced by: 1. taking Pap smears after correction of atrophy and inflammation; 2. more scrupulous colposcopy aimed at reducing the number of unsatisfactory colposcopies or misinterpreted colposcopic findings; this thorough examination should include the vagina and vulva; 3. confirmation of smear and colposcopic findings by biopsy prior to cold-knife conisation and performing a large loop excision of the transformation zone (LLETZ) for cases where there is a discrepancy between the smear abnormality and colposcopy/biopsy findings; 4. good quality cone biopsies using a technique that does not handle the mucosa and is performed after the mucosa has had time to regenerate following the colposcopic investigations; and 5. exhausting all blocks with multiple levels before reporting a cone biopsy as negative. INTRODUCTIONThe traditional management of preinvasive disease of the cervix begins with an abnormal smear detected on routine screening. The patient is referred to a colposcopist who attempts to grade, delineate and biopsy the abnormality. A colposcopic-directed biopsy confirms the smear and colposcopic diagnoses and, at a second visit, the abnormal transformation zone is destroyed. Where there is suspicion of invasion or adenocarcinoma in situ or an unsatisfactory colposcopy (defined as an inability to see the full transformation zone, the endocervical limits of a lesion or a discrepancy between the smear and colposcopic findings) a cone biopsy is performed. The cone biopsy usually provides a definitive diagnosis, with type, grade, extent of the lesion and state of the resection lines. Not infrequently, however, no lesion is found on histological examination of the cone biops...

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. Genetic variants in the complement factor H (CFH) gene are associated with AMD, but the functional consequences of many of these variants are currently unknown. In this study we aimed to determine the effect of 64 rare and low frequency variants in the CFH gene on systemic levels of factor H (FH) and complement activation marker C3bBbP using plasma samples of 252 carriers and 159 non-carriers. Individuals carrying a heterozygous nonsense, frameshift or missense variant in CFH presented with significantly decreased FH levels, and significantly increased C3bBbP levels in plasma compared to non-carrier controls. FH and C3bBbP plasma levels were relatively stable over time in samples collected during follow-up visits. Decreased FH and increased C3bBbP concentrations were observed in carriers compared to non-carriers of CFH variants among different AMD stages, with the exception of C3bBbP levels in advanced AMD stages, which were equally high in carriers and non-carriers. In AMD families, FH levels were decreased in carriers compared to non-carriers, but C3bBbP levels did not differ. Rare variants in the CFH gene can lead to reduced FH levels or reduced FH function as measured by increased C3bBbP levels. The effects of individual variants in the CFH gene reported in this study will improve the interpretation of rare and low frequency variants observed in AMD patients in clinical practice.

Complement factor I (FI) is a central inhibitor of the complement system, and impaired FI function increases complement activation, contributing to diseases such as age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (aHUS). Genetic variation in complement factor I (CFI) has been identified in both AMD and aHUS, with more than half of these variants leading to reduced FI secretion levels. For many of the variants with normal FI secretion, however, functional implications are not yet known. Here we studied 11 rare missense variants, with FI secretion levels comparable to wildtype, but a predicted damaging effects based on the Combined Annotation Dependent Depletion (CADD) score. Three variants (p.Pro50Ala, p.Arg339Gln, and p.Ser570Thr) were analyzed in plasma and serum samples of carriers affected by AMD. All 11 variants (nine for the first time in this study) were recombinantly expressed and the ability to degrade C3b was studied with the C3b degradation assay. The amount of degradation was determined by measuring the degradation product iC3b with ELISA. Eight of 11 (73%) mutant proteins (p.Pro50Ala, p.Arg339Gln, p.Ile340Thr, p.Gly342Glu, p.Gly349Arg, p.Arg474Gln, p.Gly487Cys, and p.Gly512Ser) showed significantly impaired C3b degradation, and were therefore classified as likely pathogenic. Our data indicate that genetic variants in CFI with a CADD score &gt;20 are likely to affect FI function, and that monitoring iC3b in a degradation assay is a useful tool to establish the pathogenicity of CFI variants in functional studies.

No abstract