Detection Rate of Pathogenic Mutations in ABCA4 Using Direct

Home , Stargardt disease

lous involvement in serpiginous-like choroiditis. Am J Ophthalmol. 2008; volved relatives) were counseled and consented accord- 146(5):761-766. 6. Vasconcelos-Santos DV, Rao PK, Davies JB, Sohn EH, Rao NA. Clinical fea- ing to the Declaration of Helsinki, and the study was ap- tures of tuberculous serpiginouslike choroiditis in contrast to classic serpigi- proved by the local ethics committee. Full ophthalmic nous choroiditis. Arch Ophthalmol. 2010;128(7):853-858. 7. Wong R, Graham E, Scoppettuolo E, Moin M, Stanford MR. Case report: ampigi- history, examination, and electrophysiology were per- nous chorioretinopathy associated with Eales disease in a patient with pre- formed. The patients’ DNA was extracted, and direct sumed tuberculosis. Retin Cases Brief Rep. 2011;5:249-250. (dideoxy/Sanger) sequencing of all ABCA4 exons and a 8. Friberg TR. Serpiginous choroiditis with branch vein occlusion and bilateral periphlebitis: case report. Arch Ophthalmol. 1988;106(5):585-586. 25–base pair flanking sequence was performed using standard protocols. The pathogenicity of all sequence variants was determined using standard software (Alamut Detection Rate of Pathogenic Mutations version 1.5; Interactive Biosoftware). Multiplex ligation- in ABCA4 Using Direct Sequencing: Clinical dependent probe amplification analysis (P151 and P152 and Research Implications kits; MRC-Holland) was used to identify copy number variants. We determined phase and segregation when possible. A confident molecular diagnosis was defined as the e set out to determine the mutation detection rate in 50 subjects referred with pos- presence of 2 or more pathogenic mutations. sible Stargardt disease (STGD) using direct W Results. Of 50 patients, 34 had a phenotype compatible sequencing of the ABCA4 gene (GenBank NM_000350). Pathogenic mutations in ABCA4 have been found to cause with STGD. Of these 34 patients, 27 (79%) had at least Stargardt disease (STGD)/fundus flavimaculatus as well 2 pathogenic mutations; 7 of the 34 patients had a single as some cases of cone-rod dystrophy, autosomal reces- mutation identified, 2 of the 34 had premature termina- sive retinitis pigmentosa, and bull’s-eye maculopathy.1,2 tion codons (stop codon and frameshift), and 5 of the Mutation detection rates reported in patients are highly 34 had known missense mutations. The identification of variable, depending on multiple factors including the phe- a single mutation supports the clinical diagnosis but is notype and mutation detection method used.3-5 Because not conclusive; however, the carrier rate is estimated to next-generation sequencing is likely to be introduced into be 1 in 35 to 1 in 50 patients, so we would not expect as clinical diagnostics, we revisited direct (Sanger) sequenc- many as one-fifth of our patients to be carriers by chance ing, currently the gold standard for mutation detection, alone. Among the 50 patients, 11 were diagnosed as hav- in a large group of patients with STGD to determine the ing STGD by other centers in the past but on current clini- number of patients receiving a confident molecular di- cal review were reclassified as shown in the Table, re- agnosis using a sequencing approach. flecting both progression of the disorder and the presence of phenocopies or misdiagnoses. In 5 of the 11 patients, Methods. Fifty patients with STGD or other possible the identification of 2 pathogenic mutations confirmed ABCA4 retinopathies were recruited. All patients (and in- the historical diagnosis and all had chorioretinal atro-

Table. Results From Direct Sequencing of the ABCA4 Gene in 50 Patients

Change 1 Change 2 Grade, Age at Macula Subject Amino Amino Onset, Flecks/ Additional No. Nucleotide Acid Nucleotide Acid Phase Segregation y Phenotype Cones/Rodsa Variants Conclusion 11AϾG M1V 2588GϾC G863A In trans Unaffected 30 STGD mϩ/0/0 R2030Q 3 PVs parents carriers 2 161GϾA C54Y 2588GϾC G863A In trans Affected 12 STGD m/0/0 0 2 PVs sibling with same mutations 3 161GϾA C54Y 5882GϾA G1961E NK NK 18 STGD m/0/0 0 2 PVs 4 634CϾT R212C 4457CϾT P1486L In trans Unaffected 17 STGD m/0/0 0 2 PVs parents carriers 5 2588GϾC G863A 4469GϾA C1490Y NK NK 48 STGD mϩ/0/1 0 2 PVs 6 2971GϾC G991R 4254-2AϾG Splice NK NK 21 STGD m/0/0 0 2 PVs 7 2971GϾC G991R 3602TϾG L1201R NK NK 18 STGD mϩϩ/NP/NP V643M Ͼ2 PVs (likely), G885E (likely), G1441D (unlikely), V2244V (highly likely) 8 3322CϾT R1108C 768GϾT V256V NK NK 13 STGD mϩϩ/1/1 0 2 PVs 9 3322CϾT R1108C 6079CϾT L2027F NK NK 26 STGD mϩ/0/0 0 2 PVs 10 3386GϾT R1129L 4469GϾA C1490Y In trans Unaffected 15 STGD mϩ/0/0 R152Q 2 PVs parents (unlikely) carriers

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Change 1 Change 2 Grade, Age at Macula Subject Amino Amino Onset, Flecks/ Additional No. Nucleotide Acid Nucleotide Acid Phase Segregation y Phenotype Cones/Rodsa Variants Conclusion 11 4139CϾT P1380L 5714 ϩ 5GϾA Splice NK NK 19 STGD m/0/0 0 2 PVs 12 4457CϾT P1486L 4457CϾT P1486L In trans Unaffected 25 STGD mϩϩ/1/1 0 2 PVs sibling carries 1 mutation 13 4537dupC Q1513fs 6391GϾA E2131K In trans Unaffected 10 STGD mϩ/0/0 R152Q in cis 2 PVs parents with carriers Q1513fs, E2131K in cis with E471K 14 6079CϾT L2027F 6079CϾT L2027F In trans Unaffected 28 STGD mϩϩ/0/0 0 2 PVs sibling carrier 15 5018 ϩ 2TϾC NA 6316CϾT R2106C In trans Affected 17 STGD m/0/1 0 2 PVs sibling with same mutations 16 3004CϾT R1002Wb 1957CϾT R653C In trans NK 16 STGD m/0/1 0 2 PVs 17 1253TϾC F418S 2588GϾC G863A NK NK 52 STGD mϩ/0/0 0 2 PVs 18 6709AϾC T2237Pb 3064GϾA E1022K In trans 2 Affected 6 STGD mϩϩ/0/0 0 2 PVs siblings with same mutations 19 5260TϾG Y1754D 4469GϾA C1490Y In trans NK 12 STGD mϩϩ/0/0 0 2 PVs 20 551CϾT S184Fb 4793CϾA A1598D NK 2 Affected 58 STGD m/NP/NP 0 2 PVs siblings with same mutations 21 550-551TCϾCG S184Rb 5882GϾA G1961E In trans Affected 25 STGD mϩϩ/0/0 0 2 PVs sibling with same mutations 22 5313-3CϾG Spliceb 5882GϾA G1961E In trans Unaffected 47 STGD m/0/1 0 2 PVs parents carriers 23 2588GϾC G863A 5461-10TϾC Disease- In trans NA 26 STGD mϩϩ/3/1 1 In cis with 2 PVs associated G863A allele, unknown mechanism 24 5537TϾC I1846T 5461-10TϾC Disease- In trans Unaffected 17 STGD mϩϩ/3/3 0 2 PVs associated son allele, carries unknown I1846T mechanism only 25 6089GϾA R2030Q 5461-10TϾC Disease- In trans Unaffected 4 STGD m/NP/NP 0 2 PVs associated sibling allele, carries unknown R2030Q mechanism 26 6730-1GϾC Spliceb 2588GϾC G863A NK NK 15 STGD NP/NP/NP 0 2 PVs 27 3291AϾT R1097Sb 3056CϾT T1019M In trans NK 9 STGD NP/NP/NP 1 In cis with 2 PVs R1097S 28 498delT L167HisfsX2b Not present NA NA NK 28 STGD m/1/1 0 1 PV 29 2345GϾA W782Xb Not present NA NA Unaffected 25 STGD m/1/1 0 1 PV mother carries mutation 30 2588GϾC G863A 4326CϾA N1442K NK NK 36 STGD mϩ/0/0 0 1 PV ϩ N1442K (unlikely) 31 2966TϾC V989A Not present NA NA NK 49 STGD m/1/1 0 1 PV

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phy on current clinical examination, consistent with pro- have mutations in other genes: CDH3 in subject 416 and gression of the disorder.5 One of the 11 patients with cho- CRX in subject 47.7 Subject 49 (with late-onset pattern rioretinal atrophy (subject 40) had a single stop codon, dystrophy) has a disease-associated allele of unknown again strongly supporting the original clinical diagno- mechanism and a missense variant of unknown signifi- sis. Six of the 11 patients did not have pathogenic mu- cance, making the molecular diagnosis in this patient un- tations in ABCA4. Two of the 6 have since been found to certain. In 3 of the 6 patients with a historical diagnosis

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Change 1 Change 2 Grade, Age at Macula Subject Amino Amino Onset, Flecks/ Additional No. Nucleotide Acid Nucleotide Acid Phase Segregation y Phenotype Cones/Rodsa Variants Conclusion 32 3364GϾA E1122K Not present NA NA Affected 42 STGD mϩϩ/0/0 0 1 PV sibling same mutation, 2 unaffecteds without 33 4469GϾA C1490Y 455GϾA R152Q NK NK 27 STGD mϩ/0/0 0 1 PV ϩ R152Q (unlikely) 34 6449GϾA C2150Y 4326CϾA N1442K In trans NK 8 STGD mϩϩ/4/1 0 1 PV ϩ N1442K (unlikely) 35 4139CϾT P1380L 4462TϾC C1488R In trans Affected 12 CRA CRA/NP/NP 0 2 PVs sibling (historical with diagnosis same of STGD) mutations 36 4577CϾT T1526M 5714 ϩ 5GϾA Splice In trans 2 13 CRA CRA/NP/NP 0 2 PVs Unaffected (historical siblings, diagnosis 1is of STGD) carrier for 5714 ϩ 5GϾA 37 6118CϾT R2040X 5714 ϩ 5GϾA Splice NK NK 19 CRA CRA/2/3 0 2 PVs (historical diagnosis of STGD) 38 4670AϾG Y1557C 4139CϾT P1380L NK Has 6 CRA CRA/4/4 0 2 PVs affected (historical siblings, diagnosis not seen of STGD) 39 5318CϾA A1773E 6079CϾT L2027F NK NK 16 CRA CRA/3/4 0 2 PVs (historical diagnosis of STGD) 40 655AϾT R219Xb Not present NA NA Affected 17 CRA CRA/3/4 0 1 PV sibling (historical with diagnosis same of STGD) mutation 41 NA NA 2828GϾA R943Q NA NA 21 Macular Macular 00 dystrophy dystrophy/ PVs ϩ (historical NP/NP R943Q diagnosis (unlikely) of STGD) 42 NA NA NA NA NA NA 5 RP (historical RP/4/3 0 0 PVs diagnosis of STGD) 43 768GϾT V256V 6319CϾT R2107C In trans Affected 7 BEM BEM/NP/NP 0 2 PVs sibling with same mutations 44 2588GϾC G863A 5461-10TϾC Disease- In trans NA 11 BEM BEM/0/0 1 In cis with 2 PVs associated G863A allele, unknown mechanism 45 5381CϾT A1794D Not present NA NK NK 19 BEM BEM/2/1 0 1 PV 46 NA NA NA NA NA NA 21 BEM BEM/NP/NP 0 0 PVs 47 4685TϾC I1562T NA NA NA Affected 41 BEM BEM/1/1 0 0 parent PVs ϩ and I1562T sibling, (unlikely) I1562 does not coseg- regate

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of STGD and atypical phenotypes (subjects 42, 48, and bull’s-eye maculopathy had 2 pathogenic mutations, 1 50), the molecular diagnosis remains unknown and fur- of the 5 had a single known missense mutation, and 2 of ther investigations are ongoing. Two of 5 patients with the 5 had no mutations, confirming that bull’s-eye macu-

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Change 1 Change 2 Grade, Age at Macula Subject Amino Amino Onset, Flecks/ Additional No. Nucleotide Acid Nucleotide Acid Phase Segregation y Phenotype Cones/Rodsa Variants Conclusion 48 NA NA NA NA NA NA 16 Vitelliform mϩϩ/0/1 0 0 PVs (historical diagnosis of STGD) 49 5461-10TϾC Disease- 1726GϾC D576H NA NA 50 Pattern mϩϩ/1/2 0 1 Splice associated dystrophy and 1 allele, (historical uncertain unknown diagnosis mechanism of STGD) 50 NA NA NA NA NA Unaffected 45 Atypical mϩϩ/0/0 0 0 PVs sibling pattern dystrophy (historical diagnosis of STGD)

Abbreviations: BEM, bull’s-eye maculopathy; CRA, chorioretinal atrophy; NA, not applicable; NK, not known; NP, not performed; PV, pathogenic variant; RP, retinitis pigmentosa; STGD, Stargardt disease. a The presence of flecks at the macula was classified as follows: m indicates flecks confined to the macula; mϩ, sparse flecks extending beyond the arcades; and mϩϩ, more extensive flecks outside the arcades. Cone and rod function was based on International Society for Clinical Electrophysiology of Vision electrophysiology including rod, standard bright white flash, cone flicker, and photopic electroretinograms. These were graded as follows: 0 indicates unaffected; 1, mild; 2, moderate; 3, moderately severe; and 4, severe. b Novel mutation.

lopathy is genetically heterogeneous. A total of 10 novel partment of Clinical Genetics, Churchill Hospital (Dr mutations were identified (Table). Ne´meth), Oxford, England. Correspondence: Dr Ne´meth, Nuffield Laboratory of Comment. A range of phenotypes can be associated Ophthalmology, Nuffield Department of Clinical Neu- with mutations in ABCA4; therefore, genetic testing is rosciences, University of Oxford, Level 6 West Wing, John important in establishing a firm diagnosis. Direct Radcliffe Hospital, Headley Way, Oxford OX3 9DU, sequencing in our cohort resulted in a confident England ([email protected]). molecular diagnosis in 79% of patients with an STGD Author Contributions: Dr Downes and Ms Packham con- phenotype, supporting its use as a diagnostic tool. By tributed equally to the work. comparison, the highest reported detection rate in a Conflict of Interest Disclosures: None reported. smaller group of patients was 68% using direct Funding/Support: This work was supported by the Ox- sequencing4 and 63.5% using arrayed primer exten- ford Partnership Comprehensive Biomedical Research sion.5 In patients with a historical diagnosis of STGD, Centre funded by the Department of Health NIHR Bio- more than 50% had mutations in ABCA4 testing but medical Research Centre Programme and by Oxford Rad- the remainder did not and will require further investi- cliffe Hospital Flexibility and Sustainability Funding. gation. Among our patients with STGD, 21% had only Disclaimer: The views expressed in this article are those a single mutation; further research is required to of the authors and not necessarily those of the Depart- determine the explanation for this. In our cohort, no ment of Health. cases could be explained by copy number variants. Additional Contributions: We thank the subjects and Sequencing identified several novel mutations and has their families for participating in this study and the staff the highest detection rate of available technologies. of the Oxford Eye Hospital for assistance with data col- Despite this, further research on the underlying lection, in particular Alexina Fantato, RGN, OND, genetic mechanisms in ABCA4 retinopathies is MBACP, and Clare Arnisson-Newgass, RGN. required. 1. Klevering BJ, Deutman AF, Maugeri A, Cremers FP, Hoyng CB. The spec- Susan M. Downes, MBChB, MD, FRCOphth trum of retinal phenotypes caused by mutations in the ABCA4 gene. Graefes Arch Clin Exp Ophthalmol. 2005;243(2):90-100. Emily Packham, DipRCPath 2. Michaelides M, Chen LL, Brantley MA Jr, et al. ABCA4 mutations and discor- Treena Cranston, BSc, DipRCPath dant ABCA4 alleles in patients and siblings with bull’s-eye maculopathy. Br J Ophthalmol. 2007;91(12):1650-1655. Penny Clouston, PhD, FRCPath 3. Allikmets R, Shroyer NF, Singh N, et al. Mutation of the Stargardt disease gene Anneke Seller, PhD, DipRCPath (ABCR) in age-related macular degeneration. Science. 1997;277(5333):1805- Andrea H. Ne´meth, BSc, MBBS, DPhil, FRCP 1807. 4. Shroyer NF, Lewis RA, Yatsenko AN, Wensel TG, Lupski JR. Cosegregation and functional analysis of mutant ABCR (ABCA4) alleles in families that mani- Author Affiliations: Oxford Eye Hospital (Dr Downes), fest both Stargardt disease and age-related macular degeneration. Hum Mol Genet. 2001;10(23):2671-2678. Nuffield Laboratory of Ophthalmology, Nuffield Depart- 5. Roberts LJ, Ramesar RS, Greenberg J. Clinical utility of the ABCR400 micro- ment of Clinical Neurosciences, University of Oxford, array: basing a genetic service on a commercial gene chip. Arch Ophthalmol. John Radcliffe Hospital (Drs Downes and Ne´meth), Ox- 2009;127(4):549-554. 6. Halford S, Holt R, Ne´meth AH, Downes SM. Homozygous deletion in CDH3 ford Regional Molecular Genetics Service (Mss Pack- and hypotrichosis with juvenile macular dystrophy. Arch Ophthalmol. 2012; ham and Cranston and Drs Clouston and Seller), and De- 130(11):1490-1492.

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©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/02/2021 7. Shanks ME, Downes SM, Copley RR, et al. Next-generation sequencing (NGS) Report of a Case. A 48-year-old man had a 21-year his- as a diagnostic tool for retinal degeneration reveals a much higher detection rate in early-onset disease [published online September 12, 2012]. Eur J Hum tory of deterioration of central vision. The original di- Genet. doi:10.1038/ejhg.2012.172. agnosis was Stargardt disease. Initial symptoms at age 17 years included photosensitivity, abnormal color vision, and central scotomata. His sister has the same phenotype and the parents were likely to be related. The pro- Homozygous Deletion in CDH3 and band and his sister both gave a history of having very fine, Hypotrichosis With Juvenile Macular sparse hair that never thickened, with a persistently vis- Dystrophy ible scalp (Figure 1A). Funduscopy in the proband re- vealed bilateral symmetrical macular degeneration with ypotrichosis associated with juvenile macular dys- sparing of the peripheral retina (Figure 1B and C). Vi- trophy (HJMD; OMIM 601553) is a rare autoso- sual acuities were 6/760 OD and 6/96 OS. Goldmann vi- H mal recessive disorder characterized by short scalp sual field testing showed bilateral central scotomata hair from birth and progressive macular degeneration. Loss (Figure 1D). Electrophysiology showed extinguished pat- of central vision usually occurs between the second and tern electroretinograms, normal scotopic responses, and fourth decades of life. Mutations in the P-cadherin gene significant reduction in amplitudes of both a and b waves (CDH3; GenBank NM_001793) were first reported to un- in the standard flash electroretinogram and photopic re- derlie HJMD by Sprecher et al1; splice, missense, and non- sponses. The electro-oculogram light rise was normal in sense mutations have since been described.2-5 both eyes.

A B

C D 120 105 90 75 60 135 70 45 60

150 50 30 40 30 165 15 20 10 18090 80 70 60 50 40 30 20 10 10 20 30 40 50 60 70 80 90 0

10 20 195 345 30 40 210 50 330 60

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Figure 1. Clinical spectrum of the patient with hypotrichosis associated with juvenile macular dystrophy. A, Sparse hair in the proband at age 48 years. B, Color fundus photograph showing macular degeneration. C, Color fundus photograph of the same eye showing degeneration confined to the macular and peripapillary regions, sparing the mid and peripheral retina. D, Left visual field showing extensive central scotoma (the findings are similar in the right eye).

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