Clinical Evaluation of 3 Families with Basal Laminar Drusen Caused by Novel Mutations in the Complement Factor H Gene

OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: JANEY L. WIGGS, MD, PhD ONLINE FIRST Clinical Evaluation of 3 Families With Basal Laminar Drusen Caused by Novel Mutations in the Complement Factor H Gene

Johannes P. H. van de Ven, MD; Camiel J. F. Boon, MD, PhD, FEBOpth; Sacha Fauser, MD; Lies H. Hoefsloot, PhD; Dzenita Smailhodzic, MD; Frederieke Schoenmaker-Koller, BSc; B. Jeroen Klevering, MD, PhD; Caroline C. W. Klaver, MD, PhD; Anneke I. den Hollander, PhD; Carel B. Hoyng, MD, PhD

Objectives: To identify novel complement factor H stage kidney disease as a result of membranoprolifera- (CFH) gene mutations and to specify the clinical char- tive glomerulonephritis type II. acteristics in patients with basal laminar drusen (BLD), a clinical subtype of age-related macular degeneration. Conclusions: The early-onset BLD phenotype can be caused by heterozygous mutations in the CFH gene. Be- Methods: Twenty-one probands with BLD were cause some patients with BLD are at risk to develop mem- included in this study. The ophthalmic examination branoproliferative glomerulonephritis type II, we rec- included nonstereoscopic 30° color fundus photogra- ommend that patients with extensive BLD undergo phy, fluorescein angiography, and high-resolution screening for renal dysfunction. spectral-domain optical coherence tomography. Renal function was tested by measurement of serum creati- Clinical Relevance: Elucidation of the clinical BLD phe- nine and urea nitrogen levels. Venous blood samples notype will facilitate identification of individuals predis- were drawn for genomic DNA, and all coding exons posed to developing disease-related comorbidity, such as and splice junctions of the CFH gene were analyzed by membranoproliferative glomerulonephritis type II. More- direct sequencing. over, with upcoming treatment modalities targeting specific components of the complement system, early iden- Results: In 3 families, we identified novel heterozygous mutations in the CFH gene: p.Ile184fsX, p.Lys204fsX, tification of patients with BLD and detection of the genetic and c.1697-17_-8del. Ten of 13 mutation carriers dis- defect become increasingly important. played the BLD phenotype with a wide variety in clinical presentation, ranging from limited macular drusen Arch Ophthalmol. 2012;130(8):1038-1047. to extensive drusen in the posterior pole as well as the Published online April 9, 2012. peripheral retina. Two patients with BLD developed end- doi:10.1001/archophthalmol.2012.265

GE-RELATED MACULAR DE- the genes encoding complement factor H Author Affiliations: generation (AMD) is the (CFH), complement factor B (CFB),3,4 Departments of Ophthalmology most common cause of ir- complement factor I (CFI),5 component 2 (Drs van de Ven, Boon, reversible vision loss in the (C2),3 and component 3 (C3)6-9 are associ- Smailhodzic, Klevering, den Hollander, and Hoyng and Western world among ated with increased risk of AMD. A particu- Ms Schoenmaker-Koller) and peopleA 65 years or older, with a preva- larly strong association has been reported Human Genetics (Drs Hoefsloot lence of advanced AMD of 12% after 80 by many studies for a nonsynonymous and den Hollander), Radboud years of age.1 Several risk factors for the single-nucleotide polymorphism in CFH that University Medical Center, development of AMD have been recog- encodes a tyrosine-to-histidine missense Nijmegen, the Netherlands; nized, including modifiable risk factors, variant at amino acid 402 (p.Tyr402His). Department of Vitreoretinal such as smoking, higher body mass in- Carriership of this variant increases the risk Surgery, Center for dex, and low dietary intake of antioxi- forAMDwithanoddsratiorangingfrom2.45 Ophthalmology, University of dants and zinc. Nonmodifiable risk fac- to 7.40 and may account for more than Cologne, Cologne, Germany tors include advancing age, female sex, 50% of the attributable risk of AMD.10-12 (Dr Fauser); and Departments white race, and a great variety of genetic The CFH protein inhibits the alterna- of Ophthalmology and 2 Epidemiology and Biostatistics, factors. tive pathway by competing with CFB in Erasmus Medical Center, Genes involved in the complement sys- binding to C3b, accelerating the decay of Rotterdam, the Netherlands tem have received heightened attention be- the alternative pathway C3 convertase and (Dr Klaver). cause single-nucleotide polymorphisms in acting as a cofactor for the factor I–medi-

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©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 ated proteolytic inactivation of C3b.13-15 By this mecha- final feature is the central geographic atrophy of the retinal pig- nism, CFH is essential to maintain complement homeo- ment epithelium, frequently observed after resolution of the stasis in plasma and to restrict complement activation on drusenoid pigment epithelial detachment or the development 25 complement activating self-surfaces such as the retinal of choroidal neovascularization (CNV). pigment epithelium. Age-related macular degeneration is characterized by RENAL FUNCTION multiple heterogeneous subtypes, with drusen as the hall- 16-21 Renal function was tested by measuring serum creatinine and mark lesions and usually the first clinical finding. Basal urea nitrogen levels. The following ranges were considered for laminar drusen (BLD), also termed cuticular drusen or early normal kidney function: 0.68 to 1.24 mg/dL for creatinine and adult onset, grouped drusen, is one of the subtypes in the 7.0 to 19.6 mg/dL for urea nitrogen. (To convert creatinine to AMD spectrum.22 The BLD phenotype shows character- micromoles per liter, multiply by 88.4, and to convert urea ni- istic innumerable, small, subretinal, raised yellow dru- trogen to millimoles per liter, multiply by 0.357.) sen that are hyperfluorescent on fluorescein angiography, resulting in a typical “stars-in-the-sky” appearance.23 MUTATION ANALYSIS The BLD phenotype is also associated with the p.Tyr402His variant in the CFH gene, with a risk allele Venous blood samples were drawn for genomic DNA extrac- frequency up to 70% vs 55% in “typical” AMD-affected tion from peripheral blood leukocytes. The DNA was ana- individuals.24 Boon and colleagues25 found an associa- lyzed for mutations in CFH (NCBI Entrez Gene NM_000186) tion of compound heterozygous variants in the CFH gene by polymerase chain reaction amplification of the 22 coding exons and splice junctions. Reactions were performed using stan- with BLD. Specific mutations and variants in the CFH gene dard protocols. (Primer sequences and polymerase chain re- are associated with a broad range of phenotypes, from action conditions are available from the authors on request.) early-onset renal diseases with high mortality rates to dis- Amplification products were purified, quantified on a 2% agarose orders limited to the eye, such as AMD.22 Some patients gel, and diluted for direct sequencing on an automated se- have concurrent renal and retinal abnormalities.26-29 It has quencer (BigDye Terminator, version 3 on a 3730 DNA ana- been postulated that the type, onset, and severity of re- lyzer; Applied Biosystems, Inc). Sequences were assembled using nal and/or retinal abnormalities show a considerable de- proprietary software (ContigExpress, Vector NTI suite, ver- gree of genotype-phenotype correlation.22 sion 10.0; InforMax, Inc). Each of the novel mutations iden- The purposes of this study were to identify novel CFH tified was validated through an independent polymerase chain gene mutations and to specify the clinical characteris- reaction and a sequencing reaction. tics in patients with BLD. RESULTS METHODS In 3 of the 21 probands, we identified novel heterozy- In this study, we included 21 probands diagnosed as having gous mutations in the CFH gene: 2 frameshift mutations AMD who were noted on initial examination to have BLD on in exon 5 and 1 splice-site mutation in the splice- fluorescein angiography and 192 ethnically matched control acceptor site of exon 12 (Figure 1). None of these CFH subjects of similar age who showed no signs of maculopathy. mutations were identified in 192 control subjects who had Informed consent was obtained from all subjects after expla- no signs of maculopathy, and no nonsense, frameshift, or nation of the nature and possible consequences of the study. splice-site mutations were identified in 369 ethnically We conducted the study in accordance with the tenets of the matched controls from our in-house exome database. Declaration of Helsinki, and it was approved by the Commit- The probands who carried a mutation in the CFH gene tee on Research Involving Human Subjects at the Radboud Uni- could not be distinguished clinically from the probands versity Nijmegen Medical Center, Nijmegen, the Netherlands. who did not carry a CFH mutation (Table 1). Of the 20 additional family members who underwent screening for OPHTHALMIC EXAMINATION the novel CFH gene mutations, 10 were shown to carry the same mutation as the proband, of whom 7 proved to Ophthalmic examination of the subjects included Early Treat- be affected by BLD (Figure 2 and Table 2). However, ment Diabetic Retinopathy Study visual acuity and slitlamp bio- microscopy after pupil dilatation. Digital nonstereoscopic 30° only the probands of the 3 families noticed visual loss color fundus photographs were taken with a digital fundus cam- before the diagnosis of BLD was established. era (Topcon TRC 50IX; Topcon Corporation). To confirm the Of all the patients carrying a mutation in CFH, 5 (in diagnosis of BLD, we performed fluorescein angiography and families A and B) were compound heterozygous for the high-resolution Fourier-domain optical coherence tomogra- novel CFH mutation together with the AMD risk allele phy using the combined confocal scanning laser ophthalmo- p.Tyr402His in the CFH gene. The other 8 patients (in scope/Fourier-domain optical coherence tomography device families A, B, and C) did not carry the p.Tyr402His risk (Spectralis; Heidelberg Engineering). In the early stages, the allele or carried it heterozygously on the same allele as diagnosis was based on fluorescein angiographic confirma- the mutation. An overview of the clinical and genetic char- tion of innumerable small drusen in the macula and/or periph- acteristics of the 3 families is given in Table 2. eral retina, giving a symmetrically distributed pattern of innumerable, scattered, uniformly sized, small (25- to 75-µm) hyperfluorescent lesions in both eyes. The occurrence of con- FAMILY A fluent (soft) drusen in the macular region and the subsequent development of a drusenoid pigment epithelial detachment are In family A, we identified the heterozygous c.550delA; considered characteristic for the later stages of this disease. A p.Ile184fsX frameshift mutation in exon 5. This muta-

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©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 |184fsX K204fsX c.1697-17_-8del Deletion DuplicationExon 5Intron 5-6 Deletion Exon 12 WTG G C T A C A A G A T T G A A G G A G A T WTA G A A A C C A A A G T G T G T G G G T A WT T A T T T T A C C T T T T T C A A G A A A MUTG G C T A C A A G T T G A A G G A G A T G MUTA G A A A C C A A C C A A A G T G T G T G MUT T T T T C A A G A A A G A G A A T G C G A

Figure 1. Sequences of heterozygous mutations detected in the CFH gene. For each CFH mutation, the chromatogram corresponding to the DNA sequence surrounding the mutation in CFH is shown. MUT indicates mutated CFH allele; WT, wild-type CFH allele.

tion occurs in the third short consensus repeat of the CFH had only a thin hyperpigmented border. Three years af- protein. ter the initial visual complaints, the proband reported in- The proband of family A (A-II:3) first noticed meta- creasing metamorphopsia and a rapid decrease in visual morphopsia and a decrease in visual acuity in both eyes acuity from 20/20 to 20/67 of the left eye due to classic at age 56 years. Ophthalmoscopy revealed extensive small CNV in the left eye. This neovascularization was treated and large confluent drusen in the posterior pole with a successfully during a period of 3 months with 3 intra- drusenoid pigment epithelial detachment in the macula vitreal injections of bevacizumab, 0.05 mL (25 mg/mL), of both eyes (Figure 3B and H). Among the affected sib- at an interval of 4 weeks, resulting in increased visual acu- lings of the proband, patient A-II:1 (aged 64 years) showed ity to 20/24 in that eye for 2 years as of the last exami- hard drusen in the midperipheral retina, mostly located nation. temporal to the fovea, whereas patient A-II:5 (aged 61 In addition to extensive BLD, patient B-II:1 was diag- years) had dense, macular, small and soft confluent dru- nosed as having end-stage membranoproliferative glo- sen (Figure 3A and C). Small hard drusen were seen in merulonephritis (MPGN) type II, also known as dense the peripheral retina of patient A-III:1 (aged 31 years) deposit disease, at age 48 years. She is currently being and A-III:2 (aged 27 years), with increasing numbers of treated with peritoneal dialysis and is a candidate for a these peripheral drusen with increasing age (Figure 3D, renal transplant in the near future. The other mutation- E, and G). Additional macular hard drusen were ob- carrying family members also underwent screening for served only in the oldest mutation carrier of the third gen- renal dysfunction but showed no abnormalities. eration (A-III:1) but to a lesser extent compared with his father (A-II:1). Patient A-III:4, the youngest mutation car- FAMILY C rier in this family (aged 18 years) had some soft drusen in the peripheral retina but no hard drusen as were found In family C, we identified 3 individuals with the hetero- in other family members carrying the c.550delA muta- zygous CFH gene mutation (c.1697-17_-8del) in the tion (Figure 3F). splice-acceptor site. This mutation is predicted to abol- ish the splice-acceptor site of exon 12 of the CFH gene FAMILY B given that the splice prediction score is reduced from 0.62 to 0 (as calculated by the splice-site prediction program In family B, we identified the heterozygous c.607- NNSPLICE, version 0.9; http://www.fruitfly.org/seq_tools 610dupCCAA; p.Lys204fsX frameshift mutation in exon /splice.html). 5. As was the case for the c.550delA mutation in family Only patient C-II:4 was affected with BLD. He re- A, this mutation also occurs in the region of the third ported a rapid decrease in visual acuity from 20/20 to short consensus repeat in the CFH protein. 20/35 and metamorphopsia of the right eye at age 55 years. Patient B-II:1, the proband of family B, first noticed Both fundi showed a pigment epithelial detachment and visual loss, associated metamorphopsia, and small cen- pigmentary changes in the macular area, together with tral scotomas in both eyes at age 47 years. The proband numerous small hard drusen in the midperipheral retina, and her affected siblings (B-II:4 and B-II:6) showed an mostly located temporal to the fovea. The right eye also equivalent BLD phenotype of innumerable macular hard showed a large area of parafoveal subretinal hemor- and soft drusen, with small hard drusen extending to- rhage (Figure 5A and B). In both eyes, fluorescein an- ward the peripheral retina that were symmetrical in both giography revealed more dense and well-circumscribed fundi (Figure 4). The hard drusen in the peripheral retina hyperfluorescent BLD than were seen during direct oph-

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Visual Acuity Patient Code/Age CFH p.Tyr402His, at Onset, y/Sex Age, y OD OS Retinal Phenotype CFH Mutation Allele 1/Allele 2a A-II:3/56/M 62 20/25 20/33 Both eyes: innumerable hard and confluent p.Ile184fsX Tyr/His soft drusen in midperipheral retina; macular drusenoid PED, surrounded by crystalline drusen B-II:1/47/F 52 20/17 20/24 Both eyes: macular drusen with barely p.Lys204fsX Tyr/His discernable (mid)peripheral drusen, clearly visualized on fluorescein angiography C-II:4/55/M 58 20/20 20/16 Both eyes: confluent soft drusen in c.1697-17_-8del Tyr/Tyr posterior pole, surrounded by hard drusen D/50/F 61 20/35 20/50 Both eyes: innumerable small drusen in None Tyr/His posterior pole E/54/F 60 20/20 20/25 Both eyes: innumerable small drusen None His/His scattered throughout retina F/49/F 54 20/25 20/25 Both eyes: confluent macular drusen, None Tyr/His drusenoid PED, patches of chorioretinal atrophy G/46/F 57 20/25 LP OD: confluent macular drusen surrounded None His/His by small drusen, drusenoid PED OS: fibrotic scar H/55/F 57 20/33 20/33 Both eyes: confluent macular drusen None Tyr/His surrounded by small drusen I/5/F8 71 20/33 20/25 OD: innumerable small drusen scattered None Tyr/His throughout retina, small classic CNV OS: innumerable small drusen scattered throughout retina, drusenoid PED J/68/M 75 20/25 20/80 OD: macular small drusen in posterior pole None His/His OS: macular small drusen in posterior pole, large classic CNV K/48/M 74 20/20 20/25 Both eyes: innumerable small drusen in None Tyr/His posterior pole L/65/F 68 20/33 20/1200 OD: confluent macular drusen surrounded None His/His by small drusen, drusenoid PED OS: central GA surrounded by small drusen M/55/M 55 20/16 20/16 Both eyes: extensive small drusen in None His/His posterior pole N/. . ./F 38 20/20 20/20 Both eyes: extensive soft drusen in None Tyr/His posterior pole, numerous small peripheral drusen O/67/F 71 20/20 20/20 Both eyes: innumerable small drusen None Tyr/His scattered throughout retina P/68/F 70 20/20 20/40 OD: innumerable small drusen in None Tyr/His midperipheral retina, classic CNV OS: innumerable small drusen in midperipheral retina Q/43/F 48 20/25 20/100 Both eyes: confluent macular drusen None Tyr/His surrounded by small drusen, drusenoid PED, patches of chorioretinal atrophy R/62/F 63 20/50 20/60 Both eyes: central GA surrounded by small None Tyr/His drusen S/. . ./M 43 20/20 20/20 Both eyes: small drusen scattered None His/His throughout retina T/45/F 52 20/33 20/50 Both eyes: innumerable small drusen None Tyr/Tyr scattered throughout retina U/63/M 75 20/25 20/25 Both eyes: central pseudovitelliform lesion None Tyr/His surrounded by innumerable small drusen

Abbreviations: CNV, choroidal neovascularization; ellipses, no visual loss reported; GA, geographic atrophy; His, histidine; LP, light perception; OD, right eye; OS, left eye; PED, pigment epithelial detachment; Tyr, tyrosine. a Tyr represents the wild-type allele, and His represents the risk allele.

thalmoscopy. In addition, the angiography revealed para- umab, 0.05 mL (25 mg/mL), during a period of 6 months, foveal occult CNV in the right eye. This patient was treated resulting in an increased and stabilized visual acuity of successfully with 4 intravitreal injections of bevaciz- 20/20 without metamorphopsia for 3 years to date.

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1 2 34567 II 64 62 61 60

|184fsX + |184fsX + |184fsX + ++ 402Y 402Y 402Y 402H 402Y 402H 402H 402Y

III 1 2 3 4 5 31 27 22 18 31

|184fsX + |184fsX + ++ |184fsX + ++ 402Y 402H 402Y 402H 402H 402Y 402Y 402Y 402H 402Y

B 1 2 I

1 234 56 II 52 51 50 47

K204fsX + ++ K204fsX + K204fsX + 402H 402Y 402H 402H 402H 402Y 402H 402H

III 1 2 3 26 21 17

++ ++ K204fsX + 402H 402Y 402H 402Y 402H 402Y

C 1 2 I

1 2 3 4 5 6 II 64 61 58 56

++ c.1697-17_-8del + c.1697-17_-8del + ++ 402H 402Y 402Y 402Y 402Y 402Y 402H 402Y

III 1 2 26 25

c.1697-17_-8del + ++ 402Y 402Y 402Y 402Y

Figure 2. Molecular genetic analyses of the CFH gene in families affected with basal laminar drusen (BLD). Squares indicate men; circles, women; slashes, deceased family members; black symbols, patients with BLD; shaded symbols, patients who display drusen but without BLD; numbers in the pedigree symbols, current age (in years); plus signs, the wild-type allele; 402H, the CFH Y402H risk allele; and 402Y, the CFH wild-type allele. Mutations are in red, risk alleles in orange, and wild-type alleles in black. A, All individuals affected by BLD were heterozygous for the p.Ile184fsX frameshift mutation with the exception of the youngest mutation carrier, who had only some soft peripheral drusen at the time of examination. B, All individuals carrying the p.Lys204fsX frameshift mutation were affected by BLD. C, Two carriers (C-II:2 and C-III:1) of the c.1697-17_-8del frameshift mutation did not display BLD.

A renal biopsy in patient C-II:4 at 27 years of age innumerable small hard drusen throughout the fundus showed MPGN type II, resembling the findings in pa- that are hyperfluorescent on fluorescein angiography, re- tient B-II:1. At age 46 years, end-stage kidney disease and sulting in a typical stars-in-the-sky appearance (J.P.H.V, subsequent renal failure necessitated a renal transplant. C.J.F.B., L.H.H., B.J.K., A.I.D., and C.B.H., unpublished At the time of the most recent ophthalmic investigation, data, January 2012). The age at onset of BLD is typically at age 58 years, there was no hematuria or proteinuria; earlier than that for regular AMD, and BLD are often ob- the serum creatinine level was 1.10 mg/dL. In the 2 other served in asymptomatic family members.25 The location carriers (C-II:2 and C-III:1) of the c.1697-17_-8del mu- and histopathological composition of BLD appear to be tation, we observed no fundus abnormalities and no signs identical to the drusen found in typical AMD.30 A com- of renal failure on blood test results. mon mechanism of drusen biogenesis is therefore likely. An association of the p.Tyr402His variant in the CFH COMMENT gene with both AMD and the subtype of BLD has been previously described and confirmed by several stud- A subgroup of approximately 10% of patients with AMD ies.10-12,24,31 In addition, Boon and coworkers25 were the are found to have BLD at the initial examination, that is, first to find pathogenic heterozygous mutations in the

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Patient Code/ Visual Acuity Age at Onset, CFH p.Tyr402His, y/Sexa Age, y OD OS Retinal Phenotype CFH Mutation Allele 1/Allele 2b A-I:2/. . ./F 83 NA NA Both eyes: normal Tyr/His A-II:1/. . ./M 64 NA NA Both eyes: mostly midperipheral hard drusen; p.Ile184fsX Tyr/Tyr some hard drusen in macular area A-II:3 (P)/56/M 62 20/25 20/33 Both eyes: innumerable hard and confluent soft p.Ile184fsX Tyr/His drusen in midperipheral retina; macular drusenoid PED surrounded by crystalline drusen A-II:5/. . ./M 61 NA NA Both eyes: macular hard and soft drusen p.Ile184fsX Tyr/His A-II:7/. . ./F 60 NA NA Both eyes: normal None Tyr/His A-III:1/. . ./M 31 20/20 20/20 Both eyes: hard drusen in macular area and p.Ile184fsX Tyr/His innumerable hard drusen in peripheral retina A-III:2/. . ./M 27 20/20 20/20 Both eyes: hard drusen in peripheral retina p.Ile184fsX Tyr/His A-III:3/. . ./M 22 20/12 20/12 Both eyes: normal None Tyr/His A-III:4/. . ./M 18 20/16 20/16 OD: normal p.Ile184fsX Tyr/Tyr OS: soft drusen in peripheral retina A-III:5/. . ./M 31 NA NA Both eyes: normal None Tyr/His B-II:1 (P)/47/F 52 20/17 20/24 Both eyes: macular drusen with barely p.Lys204fsX Tyr/His discernable midperipheral drusen, clearly visualized on fluorescein angiography B-II:3/. . ./F 51 20/125 (T) 20/19 OD: hyperpigmented macular scar None His/His OS: normal B-II:4/. . ./M 50 20/21 20/16 Both eyes: extensive drusen in posterior pole, p.Lys204fsX Tyr/His innumerable hard drusen in periphery B-II:6/. . ./F 47 20/26 20/16 Both eyes: mostly soft drusen in posterior pole p.Lys204fsX His/His and extensive hard drusen in peripheral retina B-III:1/. . ./F 26 20/20 20/20 Both eyes: normal None Tyr/His B-III:2/. . ./M 21 Both eyes: normal None Tyr/His B-III:3/. . ./F 17 20/13 20/17 Both eyes: soft drusen in posterior pole with p.Lys204fsX Tyr/His some subtle hard drusen in peripheral retina C-II:1/. . ./M 64 20/20 20/13 Both eyes: normal None Tyr/His C-II:2/. . ./M 61 20/25 20/20 Both eyes: normal c.1697-17_-8del Tyr/Tyr C-II:4 (P)/55/M 58 20/20 20/16 Both eyes: confluent soft drusen in posterior c.1697-17_-8del Tyr/Tyr pole, surrounded by hard drusen C-II:6/. . ./F 56 20/12 20/60 (A) Both eyes: normal None Tyr/His C-III:1/. . ./M 26 20/16 20/16 Both eyes: normal c.1697-17_-8del Tyr/Tyr C-III:2/. . ./M 25 20/20 20/20 Both eyes: normal None Tyr/Tyr

Abbreviations: A, amblyopia; ellipses, no visual loss reported; His, histidine; NA, no visual acuity available; OD, right eye; OS, left eye; P, proband; PED, pigment epithelial detachment; T, old Toxoplasma gondii infection; Tyr, tyrosine. a Probands are indicated parenthetically after the patient code. b Tyr represents the wild-type allele, and His represents the risk allele

CFH gene in association with the BLD phenotype. In ber of family A (A-III:4), who carries a CFH gene mutation, their study, the development of BLD in individuals who showed only peripheral soft drusen without the typical carry a CFH mutation on one allele in combination with hard drusen seen in patients with BLD. Because the for- the presence of the p.Tyr402His variant on the other al- mation of drusen is related to age, this patient may de- lele is described. We confirm this disease-causing velop more drusen in the future in accordance with the model by heterozygous CFH gene mutations in a sub- BLD phenotype. In family C, 1 individual of the 3 mu- group of patients affected with BLD. However, the tation carriers was affected, suggesting reduced pen- mode of inheritance of these mutations was not appar- etrance of the CFH mutation or digenic/multigenic in- ent in any of the families. Our study was not consis- heritance of variants in other genes. Alternatively, it is tently in accordance with the suggested disease model possible that a combination of genetic and acquired de- of compound heterozygosity with the p.Tyr402His vari- fects in the complement system may cause the disease, ant25 because 5 of 10 patients did not carry the CFH mu- as has been demonstrated for MPGN.32,33 tation in association with the p.Tyr402His variant on Together with a previous report on BLD caused by CFH the other allele. However, we cannot exclude that het- gene mutations,25 our findings suggest that only pa- erozygous CFH mutations will cause BLD and/or tients having specific gene mutations will develop this MPGN type II only when coinherited with as-yet un- clinical phenotype of BLD or have a greater genetic pre- identified variants in other genes. disposition to develop BLD. This is in contrast to typical The segregation of mutations in families A and B ap- AMD, which is a multifactorial disorder caused by ac- pears to be consistent with an autosomal dominant in- cumulating genetic and environmental risk.3-8,11,34 This heritance pattern. At age 18 years, the youngest mem- also might be a plausible explanation for the earlier on-

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D E F

G H

Figure 3. Retinal phenotypes of patients carrying the CFH p.Ile184fsX frameshift mutation. Fundus photography of the right eyes showed extensive hard drusen in midperipheral retina, mostly located temporally in patient A-II:1 (A); extensive soft, hard, and crystalline drusen scattered throughout the fundus in patient A-II:3 (B); and macular hard and soft drusen in patient A-II:5 (C). The green line indicates the optical coherence tomography section. Clustered groups of hard drusen (white arrowheads) were seen in the peripheral retina of patient A-III:1 (D) and patient A-III:2 (E) by fundus photography. In patient A-III:4, fundus photography showed soft drusen in the peripheral retina (F). Fluorescein angiography of the right eye of patient A-III:1 revealed more tiny hyperfluorescent drusen (G) than the number seen on color photography (D) in the peripheral retina. Optical coherence tomography (oblique section) of patient A-II:2 showed small dome-shaped elevations of the retinal pigment epithelium (H).

set of BLD compared with typical AMD. In our study, the type II and BLD.38 Because of the relatively late onset of 10 affected individuals with BLD who carried mutations MPGN type II in the 2 patients (B-II:1 and C-II:4) of our in the CFH gene showed a heterogeneous clinical pre- families, we reason that single heterozygous mutations sentation. A robust genotype-phenotype correlation of in CFH may cause late-onset MPGN type II. Given that the severity of the disease is therefore not possible be- patient B-II:1 had early-onset BLD at the initial exami- cause only the identified CFH mutations were taken into nation before renal disease was diagnosed, we recom- consideration. mend that patients with extensive early-onset BLD un- Besides BLD, specific mutations in the CFH gene can dergo screening for renal dysfunction. Despite urea and also cause MPGN type II (dense deposit disease).35 How- creatinine clearance within reference limits, MPGN and ever, the mutations we describe in this study are novel future renal dysfunction might develop because MPGN and, to our knowledge, have never been identified in pa- may be at a subclinical stage.39 tients with MPGN type II. To date, only 9 patients with Fundus changes in patients with MPGN type II vary MPGN type II have been reported to carry CFH muta- from pigmentary changes and BLD to larger soft drusen tions, and nearly all of them were homozygous or com- and CNV, finally leading to visual loss.27,28,40,41 The 2 cases pound heterozygous for missense mutations in CFH.36,37 reported in our study are the second and third reported Only 1 patient was reported to carry a single heterozy- in the literature who developed a triad of MPGN type II, gous missense mutation and to develop late-onset MPGN BLD, and CNV caused by a specific mutation in CFH.38

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D E

Figure 4. Retinal phenotypes of patients carrying the CFH p.Lys204fsX frameshift mutation. Fundus photography of the right eyes showed extensive drusen in the posterior pole extending to the peripheral retina of patients B-II:1 (A), B-II:4 (B), and B-II:6 (C). The green line indicates the optical coherence tomography section. Fluorescein angiography of patient B-II:4 showed similar but more numerous lesions (D) compared with color photography (B). Optical coherence tomography (oblique section) showed small dome-shaped elevations of the retinal pigment epithelium (E).

A B C

D E

Figure 5. Retinal phenotype patient C-II:4, carrier of the CFH c.1697-17_-8del splice-acceptor site mutation. Fundus photography of the right eye showed, besides the extensive drusen in the posterior pole, a subretinal hemorrhage (A), which is clearly visualized with fluorescein angiography at age 55 years (B). At age 58 years, fundus photography showed large, soft, confluent macular drusen surrounded by many hard drusen in the right eye (C). Fluorescein angiography at age 58 years showed densely packed hyperfluorescent drusen in the posterior pole of the right eye (D). Optical coherence tomography (oblique section) showed the density of the drusen by the dome-shaped elevations of the retinal pigment epithelium (E).

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©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 In both cases, the CNV was effectively treated with in- 5. Fagerness JA, Maller JB, Neale BM, Reynolds RC, Daly MJ, Seddon JM. Varia- travitreal injections of bevacizumab. tion near complement factor I is associated with risk of advanced AMD. Eur J Hum Genet. 2009;17(1):100-104. With upcoming treatment modalities to target spe- 6. Maller JB, Fagerness JA, Reynolds RC, Neale BM, Daly MJ, Seddon JM. Varia- cific components of the complement system, early iden- tion in complement factor 3 is associated with risk of age-related macular tification of the BLD subgroup of patients with AMD be- degeneration. Nat Genet. 2007;39(10):1200-1201. comes relevant. The strong association of this group of 7. Park KH, Fridley BL, Ryu E, Tosakulwong N, Edwards AO. Complement compo- patients with complement abnormalities may translate nent 3 (C3) haplotypes and risk of advanced age-related macular degeneration. Invest Ophthalmol Vis Sci. 2009;50(7):3386-3393. into a better response to complement-blocking therapy 8. Spencer KL, Olson LM, Anderson BM, et al. C3 R102G polymorphism increases than among patients with AMD in general. Treatment with risk of age-related macular degeneration. Hum Mol Genet. 2008;17(12):1821- a humanized monoclonal antibody that blocks comple- 1824. ment activity was shown to be successful in a patient with 9. Yates JR, Sepp T, Matharu BK, et al; Genetic Factors in AMD Study Group. 42-46 Complement C3 variant and the risk of age-related macular degeneration. N Engl atypical hemolytic uremic syndrome. In 30% of all J Med. 2007;357(6):553-561. patients with atypical hemolytic uremic syndrome, CFH 10. Edwards AO, Ritter R III, Abel KJ, Manning A, Panhuysen C, Farrer LA. Comple- gene mutations are the cause of the disease. This mani- ment factor H polymorphism and age-related macular degeneration. Science. 2005; fests as a loss of function of CFH, resulting in increased 308(5720):421-424. activity of the complement system’s alternative path- 11. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases 47-49 the risk of age-related macular degeneration. Science. 2005;308(5720):419- way. Humanized monoclonal antibodies can inhibit 421. the overactivated complement system. For this reason, 12. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age- humanized monoclonal antibody seems to be a rational related macular degeneration. Science. 2005;308(5720):385-389. candidate treatment for patients with BLD caused by mu- 13. Pangburn MK, Schreiber RD, Mu¨ller-Eberhard HJ. Human complement C3b inac- tations in the CFH gene. tivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution. J Exp Med. In summary, our findings confirm the important role 1977;146(1):257-270. of heterozygous mutations in the CFH gene in the de- 14. Weiler JM, Daha MR, Austen KF, Fearon DT. Control of the amplification con- velopment of BLD. The genotype-phenotype correla- vertase of complement by the plasma protein beta1H. Proc Natl Acad Sci U S A. tion is not straightforward, and other genetic and pos- 1976;73(9):3268-3272. sibly environmental factors may contribute to the 15. Whaley K, Ruddy S. Modulation of the alternative complement pathways by beta 1 H globulin. J Exp Med. 1976;144(5):1147-1163. development or severity of the disease. We recommend 16. Bird AC, Bressler NM, Bressler SB, et al; The International ARM Epidemiological monitoring the renal function in patients with exten- Study Group. An international classification and grading system for age-related sive BLD because some of these patients may develop maculopathy and age-related macular degeneration. Surv Ophthalmol. 1995; MPGN type II. Conversely, ophthalmic screening for BLD 39(5):367-374. 17. Gotoh N, Yamada R, Nakanishi H, et al. Correlation between CFH Y402H and HTRA1 in patients with MPGN type II is recommended because rs11200638 genotype to typical exudative age-related macular degeneration and of the risk of developing CNV and/or geographic atro- polypoidal choroidal vasculopathy phenotype in the Japanese population. Clin phy. The association of heterozygous CFH mutations and Experiment Ophthalmol. 2008;36(5):437-442. presumed ensuing complement dysfunction in patients 18. Hayashi H, Yamashiro K, Gotoh N, et al. CFH and ARMS2 variations in age- with AMD who also have BLD provides us with a prom- related macular degeneration, polypoidal choroidal vasculopathy, and retinal an- giomatous proliferation. Invest Ophthalmol Vis Sci. 2010;51(11):5914-5919. ising target for future treatments. 19. Lee KY, Vithana EN, Mathur R, et al. Association analysis of CFH, C2, BF, and HTRA1 gene polymorphisms in Chinese patients with polypoidal choroidal vasculopathy. Invest Ophthalmol Vis Sci. 2008;49(6):2613-2619. Submitted for Publication: July 14, 2011; final revision 20. Lima LH, Schubert C, Ferrara DC, et al. Three major loci involved in age-related received December 19, 2011; accepted January 26, 2012. macular degeneration are also associated with polypoidal choroidal vasculopathy. Published Online: April 9, 2012. doi:10.1001 Ophthalmology. 2010;117(8):1567-1570. /archophthalmol.2012.265 21. Mori K, Horie-Inoue K, Gehlbach PL, et al. Phenotype and genotype character- istics of age-related macular degeneration in a Japanese population. Ophthalmology. Correspondence: Johannes P. H. van de Ven, MD, De- 2010;117(5):928-938. partment of Ophthalmology, Radboud University Medi- 22. Boon CJF, van de Kar NC, Klevering BJ, et al. The spectrum of phenotypes caused cal Center, Philips van Leydenlaan, 6525 EX Nijmegen, by variants in the CFH gene. Mol Immunol. 2009;46(8-9):1573-1594. the Netherlands ([email protected]). 23. Gass JDM. Stereoscopic Atlas of Macular Disease: Diagnosis and Treatment. 2nd Financial Disclosure: None reported. ed. St Louis, MO: Mosby–Year Book; 1977. 24. Grassi MA, Folk JC, Scheetz TE, Taylor CM, Sheffield VC, Stone EM. Comple- Funding/Support: This study was supported by grant ment factor H polymorphism p.Tyr402His and cuticular drusen. Arch Ophthalmol. 016.096.309 from the Netherlands Organization for Sci- 2007;125(1):93-97. entific Research. 25. Boon CJF, Klevering BJ, Hoyng CB, et al. Basal laminar drusen caused by compound heterozygous variants in the CFH gene. Am J Hum Genet. 2008;82(2): 516-523. REFERENCES 26. Klein R, Knudtson MD, Lee KE, Klein BE. Serum cystatin C level, kidney disease markers, and incidence of age-related macular degeneration: the Beaver Dam 1. Friedman DS, O’Colmain BJ, Mun˜oz B, et al; Eye Diseases Prevalence Research Eye Study. Arch Ophthalmol. 2009;127(2):193-199. Group. Prevalence of age-related macular degeneration in the United States. Arch 27. Leys A, Proesmans W, Van Damme-Lombaerts R, Van Damme B. Specific eye Ophthalmol. 2004;122(4):564-572. fundus lesions in type II membranoproliferative glomerulonephritis. Pediatr Nephrol. 2. Jager RD, Mieler WF, Miller JW. Age-related macular degeneration. N Engl J Med. 1991;5(2):189-192. 2008;358(24):2606-2617. 28. Leys A, Vanrenterghem Y, Van Damme B, Snyers B, Pirson Y, Leys M. Sequen- 3. Gold B, Merriam JE, Zernant J, et al; AMD Genetics Clinical Study Group. Varia- tial observation of fundus changes in patients with long standing membrano- tion in factor B (BF) and complement component 2 (C2) genes is associated with proliferative glomerulonephritis type II (MPGN type II). Eur J Ophthalmol. 1991; age-related macular degeneration. Nat Genet. 2006;38(4):458-462. 1(1):17-22. 4. Spencer KL, Hauser MA, Olson LM, et al. Protective effect of complement factor 29. Nitsch D, Evans J, Roderick PJ, Smeeth L, Fletcher AE. Associations between B and complement component 2 variants in age-related macular degeneration. chronic kidney disease and age-related macular degeneration. Ophthalmic Hum Mol Genet. 2007;16(16):1986-1992. Epidemiol. 2009;16(3):181-186.

ARCH OPHTHALMOL / VOL 130 (NO. 8), AUG 2012 WWW.ARCHOPHTHALMOL.COM 1046

©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 30. Russell SR, Mullins RF, Schneider BL, Hageman GS. Location, substructure, and 40. Leys A, Vanrenterghem Y, Van Damme B, Snyers B, Pirson Y, Leys M. Fundus composition of basal laminar drusen compared with drusen associated with ag- changes in membranoproliferative glomerulonephritis type II: a fluorescein an- ing and age-related macular degeneration. Am J Ophthalmol. 2000;129(2): giographic study of 23 patients. Graefes Arch Clin Exp Ophthalmol. 1991;229 205-214. (5):406-410. 31. Despriet DD, Klaver CC, Witteman JC, et al. Complement factor H polymor- 41. McAvoy CE, Silvestri G. Retinal changes associated with type 2 glomerulonephritis. phism, complement activators, and risk of age-related macular degeneration. JAMA. Eye (Lond). 2005;19(9):985-989. 2006;296(3):301-309. 42. Chaˆtelet V, Lobbedez T, Fre´meaux-Bacchi V, Ficheux M, Ryckelynck JP, Hurault 32. Leroy V, Fremeaux-Bacchi V, Peuchmaur M, et al. Membranoproliferative glo- de Ligny B. Eculizumab: safety and efficacy after 17 months of treatment in a merulonephritis with C3NeF and genetic complement dysregulation. Pediatr renal transplant patient with recurrent atypical hemolytic-uremic syndrome: case Nephrol. 2011;26(3):419-424. report. Transplant Proc. 2010;42(10):4353-4355. 33. Licht C, Fremeaux-Bacchi V. Hereditary and acquired complement dysregula- 43. Gruppo RA, Rother RP. Eculizumab for congenital atypical hemolytic-uremic tion in membranoproliferative glomerulonephritis. Thromb Haemost. 2009; syndrome. N Engl J Med. 2009;360(5):544-546. 101(2):271-278. 44. Ko¨se O, Zimmerhackl LB, Jungraithmayr T, Mache C, Nu¨rnberger J. New treat- 34. Klein R, Peto T, Bird A, Vannewkirk MR. The epidemiology of age-related macu- ment options for atypical hemolytic uremic syndrome with the complement in- lar degeneration. Am J Ophthalmol. 2004;137(3):486-495. hibitor eculizumab. Semin Thromb Hemost. 2010;36(6):669-672. 35. Abrera-Abeleda MA, Nishimura C, Smith JL, et al. Variations in the complement 45. Mache CJ, Acham-Roschitz B, Fre´meaux-Bacchi V, et al. Complement inhibitor regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated eculizumab in atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol. 2009; with membranoproliferative glomerulonephritis type II (dense deposit disease). 4(8):1312-1316. J Med Genet. 2006;43(7):582-589. 46. Nu¨rnberger J, Philipp T, Witzke O, et al. Eculizumab for atypical hemolytic-uremic 36. Dragon-Durey MA, Fre´meaux-Bacchi V, Loirat C, et al. Heterozygous and homozygous factor H deficiencies associated with hemolytic uremic syndrome or mem- syndrome [published correction appears in N Engl J Med. 2009;360(23):2487]. branoproliferative glomerulonephritis: report and genetic analysis of 16 cases. N Engl J Med. 2009;360(5):542-544. J Am Soc Nephrol. 2004;15(3):787-795. 47. Bu¨ttner-Mainik A, Parsons J, Je´roˆme H, et al. Production of biologically active 37. Zipfel PF, Heinen S, Jo´zsi M, Skerka C. Complement and diseases: defective al- recombinant human factor H in Physcomitrella. Plant Biotechnol J. 2011;9 ternative pathway control results in kidney and eye diseases. Mol Immunol. 2006; (3):373-383. 43(1-2):97-106. 48. Caprioli J, Noris M, Brioschi S, et al; International Registry of Recurrent and Fa- 38. Montes T, Goicoechea de Jorge E, Ramos R, et al. Genetic deficiency of comple- milial HUS/TTP. Genetics of HUS: the impact of MCP, CFH, and IF mutations on ment factor H in a patient with age-related macular degeneration and membrano- clinical presentation, response to treatment, and outcome. Blood. 2006;108 proliferative glomerulonephritis. Mol Immunol. 2008;45(10):2897-2904. (4):1267-1279. 39. Lorenz EC, Sethi S, Leung N, Dispenzieri A, Fervenza FC, Cosio FG. Recurrent 49. Rohrer B, Long Q, Coughlin B, et al. A targeted inhibitor of the complement al- membranoproliferative glomerulonephritis after kidney transplantation. Kidney ternative pathway reduces RPE injury and angiogenesis in models of age- Int. 2010;77(8):721-728. related macular degeneration. Adv Exp Med Biol. 2010;703:137-149.

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