Loss-of-Function Mutations in the CFH Gene Affecting Alternatively Encoded Factor H-like 1 Protein Cause Dominant Early-Onset Macular Drusen
Authors: Rachel L. Taylor, James A. Poulter, Susan M. Downes, Martin McKibbin, Kamron N. Khan, Chris F. Inglehearn, Andrew R. Webster, Alison J. Hardcastle, Michel Michaelides, Paul N. Bishop, Simon J. Clark, Graeme C. Black, Graeme Black, Georgina Hall, Stuart Ingram, Rachel Taylor, Forbes Manson, Panagiotis Sergouniotis, Andrew Webster, Alison Hardcastle, Michel Michaelides, Vincent Plagnol, Nikolas Pontikos, Michael Cheetham, Gavin Arno, Alessia Fiorentino, Chris Inglehearn, Carmel Toomes, Manir Ali, Martin McKibbin, Claire Smith, Kamron Khan, Susan Downes, Jing Yu, Stephanie Halford, Suzanne Broadgate, Veronica van Heyningen
Journal: Ophthalmology (2019)

PurposeTo characterize the molecular mechanism underpinning early-onset macular drusen (EOMD), a phenotypically severe subtype of age-related macular degeneration (AMD), in a subgroup of patients.DesignMulticenter case series, in vitro experimentation, and retrospective analysis of previously reported variants.ParticipantsSeven families with apparently autosomal dominant EOMD.MethodsPatients underwent a comprehensive ophthalmic assessment. Affected individuals from families A, B, and E underwent whole exome sequencing. The probands from families C, D, F, and G underwent Sanger sequencing analysis of the complement factor H (CFH) gene. Mutant recombinant factor H like-1 (FHL-1) proteins were expressed in HEK293 cells to assess the impact on FHL-1 expression and function. Previously reported EOMD-causing variants in CFH were reviewed.Main Outcome MeasuresDetailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD.ResultsAll affected participants demonstrated bilateral drusen. The earliest reported age of onset was 16 years (median, 46 years). Ultra-rare (minor allele frequency [MAF], ≤0.0001) CFH variants were identified as the cause of disease in each family: CFH c.1243del, p.(Ala415ProfsTer39) het; c.350+1G→T het; c.619+1G→A het, c.380G→A, p.(Arg127His) het; c.694C→T p.(Arg232Ter) het (identified in 2 unrelated families in this cohort); and c.1291T→A, p.(Cys431Ser). All mutations affect complement control protein domains 2 through 7, and thus are predicted to impact both FHL-1, the predominant isoform in Bruch's membrane (BrM) of the macula, and factor H (FH). In vitro analysis of recombinant proteins FHL-1R127H, FHL-1A415f/s, and FHL-1C431S demonstrated that they are not secreted, and thus are loss-of-function proteins. Review of 29 previously reported EOMD-causing mutations found that 75.8% (22/29) impact FHL-1 and FH. In total, 86.2% (25/29) of EOMD-associated variants cause haploinsufficiency of FH or FHL-1.ConclusionsEarly-onset macular drusen is an underrecognized, phenotypically severe subtype of AMD. We propose that haploinsufficiency of FHL-1, the main regulator of the complement pathway in BrM, where drusen develop, is an important mechanism underpinning the development of EOMD in a number of cases. Understanding the molecular basis of EOMD will shed light on AMD pathogenesis given their pathologic similarities. To characterize the molecular mechanism underpinning early-onset macular drusen (EOMD), a phenotypically severe subtype of age-related macular degeneration (AMD), in a subgroup of patients. Multicenter case series, in vitro experimentation, and retrospective analysis of previously reported variants. Seven families with apparently autosomal dominant EOMD. Patients underwent a comprehensive ophthalmic assessment. Affected individuals from families A, B, and E underwent whole exome sequencing. The probands from families C, D, F, and G underwent Sanger sequencing analysis of the complement factor H (CFH) gene. Mutant recombinant factor H like-1 (FHL-1) proteins were expressed in HEK293 cells to assess the impact on FHL-1 expression and function. Previously reported EOMD-causing variants in CFH were reviewed. Detailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD. All affected participants demonstrated bilateral drusen. The earliest reported age of onset was 16 years (median, 46 years). Ultra-rare (minor allele frequency [MAF], ≤0.0001) CFH variants were identified as the cause of disease in each family: CFH c.1243del, p.(Ala415ProfsTer39) het; c.350+1G→T het; c.619+1G→A het, c.380G→A, p.(Arg127His) het; c.694C→T p.(Arg232Ter) het (identified in 2 unrelated families in this cohort); and c.1291T→A, p.(Cys431Ser). All mutations affect complement control protein domains 2 through 7, and thus are predicted to impact both FHL-1, the predominant isoform in Bruch's membrane (BrM) of the macula, and factor H (FH). In vitro analysis of recombinant proteins FHL-1R127H, FHL-1A415f/s, and FHL-1C431S demonstrated that they are not secreted, and thus are loss-of-function proteins. Review of 29 previously reported EOMD-causing mutations found that 75.8% (22/29) impact FHL-1 and FH. In total, 86.2% (25/29) of EOMD-associated variants cause haploinsufficiency of FH or FHL-1. Early-onset macular drusen is an underrecognized, phenotypically severe subtype of AMD. We propose that haploinsufficiency of FHL-1, the main regulator of the complement pathway in BrM, where drusen develop, is an important mechanism underpinning the development of EOMD in a number of cases. Understanding the molecular basis of EOMD will shed light on AMD pathogenesis given their pathologic similarities.