OPHTHALMIC MOLECULAR GENETICS Genotype-Phenotype Correlation for Leber Congenital Amaurosis in Northern Pakistan

Martin McKibbin, FRCOphth; Manir Ali, PhD; Moin D. Mohamed, FRCS; Adam P. Booth, FRCOphth; Fiona Bishop, FRCOphth; Bishwanath Pal, FRCOphth; Kelly Springell, BSc; Yasmin Raashid, FRCOG; Hussain Jafri, MBA; Chris F. Inglehearn, PhD

Objectives: To report the genetic basis of Leber con- in predicting the genotype. Many of the phenotypic vari- genital amaurosis (LCA) in northern Pakistan and to de- ables became more prevalent with increasing age. scribe the phenotype. Conclusions: Leber congenital amaurosis in northern Methods: DNA from 14 families was analyzed using single- Pakistan is genetically heterogeneous. Mutations in RP- nucleotide polymorphism and microsatellite genotyping GRIP1, AIPL1, and LCA5 accounted for disease in 10 of and direct sequencing to determine the genes and muta- the 14 families. This study illustrates the differences in tions involved. The history and examination findings from phenotype, for both the anterior and posterior seg- 64 affected individuals were analyzed to show genotype- ments, seen between patients with identical or different phenotype correlation and phenotypic progression. mutations in the LCA genes and also suggests that at least some of the phenotypic variation is age dependent. Results: Homozygous mutations were found in RPGRIP1 (4 families), AIPL1 and LCA5 (3 families each), and RPE65, Clinical Relevance: The LCA phenotype, especially one CRB1, and TULP1 (1 family each). Six of the mutations including different generations in the same family, may are novel. An additional family demonstrated linkage to be used to refine a molecular diagnostic strategy. the LCA9 locus. Visual acuity, severe keratoconus, cata- ract, and macular atrophy were the most helpful features Arch Ophthalmol. 2010;128(1):107-113

EBER CONGENITAL AMAURO- notype of 64 affected individuals and re- sis (LCA) is the earliest and veal differences in the frequency of key phe- most severe form of retinal notypic features between individuals with dystrophy. It usually mani- mutations in different genes and evidence fests at or shortly after birth of progression with increasing age. withL severely impaired vision, amaurotic pupils, and sensory nystagmus. At pres- METHODS ent, 14 genes or loci have been impli- cated in the pathogenesis and these en- code proteins involved in developmental We examined 55 families with a variety of in- Author Affiliations: and physiological pathways in the retina.1 herited eye diseases in northern Pakistan. Af- Department of Ophthalmology, Despite the advent of genotyping microar- ter the initial examination, members of each St. James’s University Hospital rays for LCA, the genetic heterogeneity of family were invited to participate in research (Drs McKibbin and Bishop), aiming to identify novel genes implicated in in- and Section of Ophthalmology this condition continues to present a sig- herited retinal degeneration. Informed con- and Neuroscience, Leeds nificant obstacle to clinicians. Knowledge sent was obtained from participants and from Institute of Molecular Medicine of genotype-phenotype correlations and of the elders in each household. This study was (Drs McKibbin, Ali, and ethnic variations in the prevalence of mu- performed according to the principles of the Inglehearn, Ms Springell, and tations in the different LCA genes would Declaration of Helsinki, using a process ap- Mr Jafri) Leeds, St Thomas’s help to refine a molecular diagnostic strat- proved by a UK Research Ethics Committee. Hospital (Dr Mohamed) and egy. Molecular characterization of LCA is A clinical diagnosis of LCA was made in 23 Moorfields Eye Hospital important for genetic counseling and gene families, based on the clinical history, reces- (Dr Pal), London, and Royal replacement may be a potential therapy.2,3 sive inheritance, and examination findings. Eye Infirmary, Plymouth In this article, we report the completed These included visual acuity testing and ante- (Dr Booth), England; and rior and posterior segment examination using Department of Obstetrics and genetic screening of 14 LCA families from direct and indirect ophthalmoscopy. Cases were Gynaecology, King Edward northern Pakistan. Our findings provide es- examined in their own community and in a Medical University, Lahore, timates of the different frequency of mu- nonmedical setting. As a result, neither ocu- Pakistan (Dr Raashid and tations in the known LCA genes in this lar electrophysiology nor corneal topography Mr Jafri). population. In addition, we review the phe- were available.

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 A B

Figure 1. Color fundus photographs of the right (A) and left (B) eyes of a 23-year-old man homozygous for the common mutation in AILP1. There is ill-defined macular atrophy in the left eye, with a well-defined staphyloma in the right eye. Nummular or coarse pigment clumping is seen at the temporal and superior edges of the staphyloma in the right eye. Peripheral bone spiculation is seen in the midperipheral retina of the right eye, below the optic disc. Optic disc drusen are present in both eyes.

Affected and unaffected cases were examined by 2 ophthal- resultant genotypes were examined using the program IBD- mologists (M.M. and either A.P.B. or M.D.M.). Visual acuity finder (http://dna.leeds.ac.uk/ibdfinder) to identify regions of ho- was recorded together with a history of nyctalopia or photoa- mozygosity shared by all affected family members. version. The presence or absence of 5 key phenotypic vari- Homozygosity at a given locus was confirmed by polymerase ables was then noted. Each variable was felt to be present when chain reaction amplifying microsatellite marker alleles from af- noted in at least 1 eye. Severe keratoconus was considered to fected and unaffected family members. Products were resolved be present when apical scarring, an “oil droplet” reflex, or a by electrophoresis on a 3130xl Genetic Analyzer (Applied Bio- positive Munson sign was noted on anterior segment exami- systems, Warrington, England). The results were analyzed using nation. No patient underwent corneal topography and so less Genemapper version 4.0 software (Applied Biosystems). severe cases may have been overlooked. Significant cataract was Any known LCA genes within regions of shared homozy- considered present when posterior subcapsular or cortical cata- gosity were sequenced to search for mutations. Primer pairs en- ract was noted or cataract surgery had been performed previ- compassing the exons of each gene were used in a polymerase ously. Subjects with lens dislocation and/or aphakia were not chain reaction to amplify products that were initially digested included in the analysis. Macular atrophy was recorded as pres- with ExoSAP-IT (GE Healthcare, Chalfont St Giles, England). ent when there was evidence of either atrophy or a staphy- This DNA was then sequenced using the BigDye Terminator loma, as have previously been noted in LCA.4 Nummular or version 3.1 Cycle Sequencing Kit (Applied Biosystems) and ana- coarse pigment clumping in the macula was recorded sepa- lyzed on a 3130xl Genetic Analyzer. rately from peripheral intraretinal pigment migration or bone spicule pigmentation (Figure 1). If an adequate examination RESULTS of the anterior or posterior segment was rendered impossible by media opacity, then the presence or absence of that finding was recorded as not known. The overall prevalence of each of GENETIC ANALYSIS the 5 key phenotypic features was compared for each of the genes and loci implicated in the pathogenesis. Each feature was noted The molecular diagnostic strategy has identified disease- to be either universal, common (present in 50%-99% of sub- causing mutations or linkage to known loci in 14 of the jects), rare (present in 1%-49%), or absent. Evidence of phe- 23 LCA families (61%) to date. These involve AIPL1 (3 notypic progression with age was provided by recording the cu- families), RGRIP1 (4 families), LCA5 (3 families), CRB1 mulative prevalence of these clinical features for all individuals younger than a certain age, measured in decades, when known. (1 family), TULP1 (1 family), RPE65 (1 family), and the DNA was extracted from peripheral blood leukocytes. Three LCA9 locus (1 family). All cases were homozygous for the different approaches were used to obtain a molecular diagnosis. relevant mutation, consistent with autozygosity at these In 3 families identified initially, a whole-genome, microsatellite- loci (Table 1). Six of these mutations are new, namely based linkage search revealed linkage to the LCA5 locus, lead- the c.587ϩ1GϾC, c.1180CϾT, and c.3620TϾG muta- ing to the identification of the gene involved.5 For families sampled tions in RPGRIP1, the c.107CϾG mutation in CRB1, the more recently, single affected DNA from each family was sent c.1138AϾG mutation in TULP1, and the c.751GϾT mu- to Asper Biotech for microarray screening on the LCA mutation tation in RPE65. In each case, the relevant alleles segre- chip available at the time (www.asperbio.com). Finally, for those gated with disease. Mutations not previously reported in families in whom no mutation was found using this microarray, the literature were tested for frequency in ethnically matched DNA from affected individuals was mixed in equimolar controls. The c.587ϩ1GϾC mutation in RPGRIP1, the amounts then screened in a single hybridization using the Af- Ͼ Ͼ fymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, c.1138A G mutation in TULP1, and the c.751G T mu- Santa Clara, California) to generate more than 1 million single- tation in RPE65 were excluded from DNA of 171 con- nucleotide polymorphism genotypes. This analysis was per- trols. The second and third of these cause nonconserva- formed by AROS Applied Biotechnology (www.arosab.com). The tive change in evolutionary conserved residues, while the

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 Table 1. Mutations and Linkage Identified in 14 Families With Leber Congenital Amaurosis From Northern Pakistan

Mutation 1 Mutation 2

Gene Family Nucleotide Change Amino Acid Change Nucleotide Change Amino Acid Change Type of Mutation AIPL1 MEP20 c.834 GϾA p.Trp278X c.834 GϾA p.Trp278X Nonsense MEP21 c.834 GϾA p.Trp278X c.834 GϾA p.Trp278X Nonsense MEP29 c.834 GϾA p.Trp278X c.834 GϾA p.Trp278X Nonsense RPGRIP1 MEP1 c.587ϩ1GϾCa Unclear c.587ϩ1GϾCa Unclear Missense MEP3 c.3620TϾGa p.Leu1207X c.3620TϾGa p.Leu1207X Nonsense MEP43 c.1180CϾTa p.Gln394X c.1180CϾTa p.Gln394X Nonsense MEP50 c.2480 GϾT p.Arg827Leu c.2480 GϾT p.Arg827Leu Missense LCA5 MEP2 c.1151delC p.Pro384GlnfsX17 c.1151delC p.Pro384GlnfsX17 Nonsense MEP4 c.1151delC p.Pro384GlnfsX17 c.1151delC p.Pro384GlnfsX17 Nonsense MEP25 c.1151delC p.Pro384GlnfsX17 c.1151delC p.Pro384GlnfsX17 Nonsense CRB1 MEP53 c.107CϾGa p.Ser36X c.107CϾGa p.Ser36X Nonsense TULP1 MEP51 c.1138AϾGa p.Thr380Ala c.1138AϾGa p.Thr380Ala Missense RPE65 MEP55 c.751GϾTa p.Val251Phe c.751GϾTa p.Val251Phe Missense LCA9-linked MEP 34

a Denotes a new mutation.

Table 2. Age, Visual Acuity, and Prevalence of Nyctalopia and Photoaversion for Each Gene or Locus

No. of Cases Age, y, Prevalence of Prevalence of Gene or Locus Examined Mean (Range) Visual Acuity Nyctalopia (%) Photoaversion (%) AIPL1 10 21.7 (7-43) HM to PL Rare (20) Rare (10) RPGRIP1 16 23.5 (8-67) 6/60 to PL Rare (38) Rare (31) LCA5 13 22.2 (2-43) 6/60 to NPL Common (92) Common (77) CRB1 10 28.6 (9-65) HM to PL Rare (10) Rare (10) TUPL1 3 27.7 (19-40) 6/76 to CF Universal (100) Absent RPE65 6 26 (17-30) 6/76 to CF Universal (100) Common (50) LCA9-linked 6 18 (13-21) PL Absent Common (83)

Abbreviations: CF, counting fingers; HM, hand movements; NPL, no perception of light; PL, perception of light.

first almost certainly ablates the splice donor site for exon or 6/76 was obtained only for the RPGRIP1, LCA5, TULP1, 4. The premature stop codon mutations, c.107CϾGinCRB1 and RPE65 phenotypes. Nyctalopia was a universal find- and c.3620TϾG and c.1180CϾTinRPGRIP1, were ex- ing in the families with RPE65- and TULP1-related dis- cluded from DNA of 48 controls, which was felt to be suf- ease. It was common in LCA5-linked disease (92% of sub- ficient given that these mutations almost certainly repre- jects), rare in AIPL1-, RPGRIP1-, and CRB1-related disease, sent complete null alleles. and absent in all cases with LCA9-linked disease. Pho- The AIPL1 c.834 GϾA and LCA5 c.1151delC muta- toaversion was a common finding in LCA5- (77% of sub- tions, each of which accounted for LCA in 3 of the 14 jects), LCA9- (83%), and RPE65-related (50%) disease. families, are almost certainly founder mutations shared It was rare in AIPL1-, RPGRIP1-, and CRB1-related dis- between distantly related families. Affected members of ease and absent in TULP1-related disease (Table 2). each family shared a common haplotype across the linked Severe keratoconus was common in cases with mu- chromosomal region. tations in CRB1 (70%) and AIPL1 (60%) but was not seen in cases with TULP1 mutations (Table 3). It was not seen PHENOTYPIC ANALYSIS BY GENE before the second decade, except in the families with AIPL1 mutations. Keratoconus became more common with in- For the 14 families in whom a disease-causing mutation or linkage was identified, the number of affected cases exam- creasing age in families with mutations in AIPL1, CRB1, ined, the mean age, and visual acuity at the time of exami- RPGRIP1, LCA5,orRPE65. The cumulative prevalence nation are given in Table 2, together with the prevalence of keratoconus is shown in Figure 2B. of nyctalopia and photoaversion. For the 5 key pheno- Cataract was a universal finding in cases with LCA9 link- typic variables, the overall prevalence for each gene/locus age and was common with AIPL1 (70%), TULP1 (67%), and is given in Figure 2A, C, E, G, and I. Table 3 gives the RPGRIP1 (54%) mutations (Table 3). It was never seen be- frequency of each variable, when known, by gene or locus. fore the second decade and was a late feature in LCA5- and CRB1-related LCA, not seen before the fifth and sixth de- PHENOTYPE-GENOTYPE CORRELATION cades, respectively. Cataract was absent from the RPE65 se- AND PROGRESSION ries. It became more prevalent with increasing age in the RPGRIP1,AIPL1,LCA5,CRB1,andTULP1series(Figure2D). Visual acuity ranged from 6/60 to no perception of light Macular atrophy was a universal finding in the LCA9- in all subjects. A recordable Snellen acuity of either 6/60 linked family and in those with mutations in AIPL1 and

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 18 100 A Not known B AIPL1 90 16 Normal RPGRIP1 80 14 Keratoconus LCA5 70 12 CRB1 60 TULP1 10 50 RPE65 8 40 LCA9 30

No. of Cases 6 20 4 10

2 Cumulative Prevalence, % 0 0 AIPL1 RPGRIP1 LCA5 CRB1 TULP1 RPE65 LCA9 <10 <20 <30 <40 <50 <60 <70 Gene or Locus Age Range, y

18 100 C Not known D AIPL1 90 16 Clear lens RPGRIP1 80 14 Cataract LCA5 70 12 CRB1 60 TULP1 10 50 RPE65 8 40 LCA9 30

No. of Cases 6 20 4 10

2 Cumulative Prevalence, % 0 0 AIPL1 RPGRIP1 LCA5 CRB1 TULP1 RPE65 LCA9 <10 <20 <30 <40 <50 <60 <70 Gene or Locus Age Range, y

18 100 E Not known F AIPL1 90 16 Normal RPGRIP1 80 14 Macular atrophy LCA5 70 12 CRB1 60 TULP1 10 50 RPE65 8 40 LCA9 30

No. of Cases 6 20 4 10

2 Cumulative Prevalence, % 0 0 AIPL1 RPGRIP1 LCA5 CRB1 TULP1 RPE65 LCA9 <10 <20 <30 <40 <50 <60 <70 Gene or Locus Age Range, y

18 100 G Not known H AIPL1 90 16 None RPGRIP1 80 14 Nummular pigment LCA5 70 12 CRB1 60 TULP1 10 50 RPE65 8 40 LCA9 30

No. of Cases 6 20 4 10

2 Cumulative Prevalence, % 0 0 AIPL1 RPGRIP1 LCA5 CRB1 TULP1 RPE65 LCA9 <10 <20 <30 <40 <50 <60 <70 Gene or Locus Age Range, y

18 100 I Not known J AIPL1 90 16 None RPGRIP1 80 14 Bone spiculation LCA5 70 12 CRB1 60 TULP1 10 50 RPE65 8 40 LCA9 30

No. of Cases 6 20 4 10

2 Cumulative Prevalence, % 0 0 AIPL1 RPGRIP1 LCA5 CRB1 TULP1 RPE65 LCA9 <10 <20 <30 <40 <50 <60 <70 Gene or Locus Age Range, y

Figure 2. Total and cumulative prevalence of severe keratoconus (A and B, respectively), cataract (C and D, respectively), macular atrophy (E and F, respectively), nummular pigmentation (G and H, respectively), and peripheral bone spiculation (I and J, respectively).

TULP1. It was a common finding with CRB1 (88%) and first decade only in the family with mutations in CRB1. LCA5 (64%) mutations. It was often seen in the first de- It was a late finding with RPGRIP1 and TULP1 muta- cade, except in families with RPGRIP1 mutations, when tions, not seen until the fourth and fifth decades, respec- it was not seen before the third decade. It was absent from tively (Figure 2H). the family with RPE65 mutations (Figure 2F). A pigmentary retinopathy was a universal finding with Nummular pigmentation was a common finding with RPE65- and LCA9-linked disease. It was a common find- RPE65- (83%), LCA5- (64%), CRB1- (71%), TULP1- (50%), ing with mutations in all the other genes. It was never and LCA9-linked (50%) disease. It was seen during the seen before the second decade and became increasingly

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 Table 3. Prevalence of the Anterior and Posterior Segment Signs for Each Gene or Locus

Prevalence of (%)

Severe Macular Nummular Bone Spicule Gene or Locus Keratoconus Cataract Atrophy Pigmentation Pigmentation AIPL1 Common (60) Common (70) Universal (100) Rare (11) Common (80) RPGRIP1 Rare (38) Common (54) Rare (31) Rare (23) Common (57) LCA5 Rare (15) Rare (8) Common (64) Common (64) Common (64) CRB1 Common (70) Rare (22) Common (88) Common (71) Common (57) TUPL1 Absent Common (67) Universal (100) Common (50) Common (67) RPE65 Rare (17) Absent Absent Common (83) Universal (100) LCA9-linked Rare (17) Universal (100) Universal (100) Common (50) Universal (100)

prevalent with increasing age in all the families in which ful in distinguishing the LCA5 phenotype from the other it was not universal (Figure 2J). commonly implicated genes.13-15 The CRB1-related phe- notype shares the late-onset cataract described in other 10,12,16 COMMENT series. However, the absence of nyctalopia, consis- tently poor acuity, frequent keratoconus, and the univer- This study confirms that LCA is genetically heteroge- sal macular atrophy and pigmentary retinopathy noted in neous, even within a population from a defined geo- this series are at odds with observations in other series of graphical area and in which consanguineous marriage is CRB1 mutations. Furthermore, none of the cases or car- common. The genes most commonly involved in this LCA riers in this series had either retinal exudation or pre- series from northern Pakistan are RPGRIP1 (29% of fami- served para-arterial retinal pigment epithelium and these features have previously been considered almost diagnos- lies with molecular confirmation), AIPL1 (21% of fami- 10,12,16-18 lies), and LCA5 (21% of families). This contrasts with tic of CRB1-related LCA. The CRB1 mutation in this pooled data from other series that identified the 3 most series is a new nonsense mutation and this may account common genes to be CEP290 (15% of cases), GUCY2D for the different phenotype. Data in the other published (12% of cases), and CRB1 (10% of cases) whereas the series cannot be easily analyzed by the class of mutation. RPGRIP1, AIPL1, and LCA5 genes accounted for only 4.2%, This series of 16 RPGRIP1-related cases is the largest 5.3%, and 1.8% of cases, respectively.1 This probably re- published to date, to our knowledge, and a comparison flects the different ethnic origins of the series studied and with other RPGRIP1 mutations is difficult given the small 8,10-12 the presence of founder mutations within these popula- numbers in most other series. The pooled pheno- tions. It also suggests that different screening strategies may type for all 4 families was different from the AIPL1- be required to obtain a molecular diagnosis in different related phenotype. Both nyctalopia and photoaversion ethnic groups, particularly when microarrays are used.1,6 were more frequent. Some patients had a recordable The Asper Biotech LCA microarray (www.asperbio.com) Snellen acuity of 6/60. The anterior segment findings and detected only 2 of the 9 mutations identified, accounting the presence of macular atrophy were less frequent and for 4 of the 14 families. These were the AIPL1 c.834GϾA were not seen before the second decade. and RPGRIP1 c.2480GϾT mutations, both published pre- For all the genes in this series, the most striking phe- viously.7,8 This contrasts with our findings in white cases notypic difference was between the RPE65-related and with LCA in which the microarray detected at least 1 al- the other phenotypes. All 6 cases with the RPE65 phe- lele for 4 of the 5 patients screened (data not shown). This notype had poor visual function with nyctalopia identi- probably reflects a bias in the literature to date toward the fied shortly after birth, but only 1 had nystagmus at the screening of white subjects with LCA as the microarray is time of examination. This was followed by photoaver- designed to detect all published LCA mutations. sion from the third decade in 3 cases, at which point acu- For 4 of the genes involved in the pathogenesis, namely ity and reading ability deteriorated rapidly. This evolu- RPGRIP1, LCA5, AIPL1, and CRB1, the number of af- tion of symptoms and the absence of many other fected individuals with homozygous mutations is suf- examination findings are in keeping with most other se- ficiently large to provide useful genotype-phenotype ries with RPE65 phenotypic data.12,13,19-21 correlation data and to compare with other series. The Having identified the genes most often implicated in AIPL1-related phenotype in this and other series is se- causing LCA in a given ethnic group, the phenotypic data vere, with poor acuity, frequent keratoconus, lens opaci- may help to refine the choice of genes screened. Hanein ties, macular atrophy, and bone spiculation, often from et al10 have previously suggested a strategy, based on a the first decade.7,9-12 However, neither nyctalopia nor pho- combination of physical signs and symptoms, to iden- toaversion were common and the anterior segment find- tify the likely causative gene. This strategy would have ings in this series were more common than has been re- been partially successful in our families. For example, nyc- ported previously. For LCA5, the high prevalence of talopia as a common finding would have implicated RPE65 nyctalopia, photoaversion, and nummular pigment, to- and TULP1, as suggested by Hanein et al, but not CRB1. gether with the low frequency of severe keratoconus and Similarly Hanein et al felt that photoaversion implicated cataract, were the features that seemed to be more help- both RPGRIP1- and AIPL1-related disease but this was

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 not the case in this series. Our experience is that nycta- the pathogenesis. However, the prevalence of many signs lopia and light sensitivity do not accurately help predict varies with age and this must be considered. the genes responsible, even when a reliable history of func- tion from birth is available, and this has been the expe- Submitted for Publication: January 31, 2009; final re- rience of others.12,18,22 Instead, we would suggest that the vision received May 5, 2009; accepted May 10, 2009. use of objective data relating to the presence of key physi- Correspondence: Martin McKibbin, FRCOphth, Depart- cal signs may be the most helpful way to implicate cer- ment of Ophthalmology, St. James’s University Hospi- tain genes and to render others as less likely to be in- tal, Beckett Street, Leeds LS9 7TF, England (martin volved in the pathogenesis.10 Given the progression of [email protected]). many phenotypic features, the age of an individual sub- Financial Disclosure: None reported. ject must be considered. In a family in which several fam- Funding/Support: This work was funded by Yorkshire ily members are affected, the use of pooled phenotypic Eye Research and grant 073477 from the Wellcome Trust. data from individuals across the age spectrum will prob- Dr Ali is funded through a Medical Research Council New ably carry the greatest predictive value. This value would Investigator Award. be made even greater if the novel databases for inherited retinal disease provided accurate and continuous rather REFERENCES than categorical phenotype data for a larger number of cases than are found in this article. 1. den Hollander AI, Roepman R, Koenekoop RK, Cremers FPM. Leber congenital Mutations in 4 of the genes in this series, namely amaurosis: genes, proteins and disease mechanisms. Prog Retin Eye Res. 2008; RPGRIP1, CRB1, TULP1, and RPE65, have been reported 27(4):391-419. 2. Bainbridge JW, Smith AJ, Barker SS, et al. Effect of gene therapy on visual func- to cause either LCA or an acquired, later-onset rod-cone tion in Leber’s congenital amaurosis. N Engl J Med. 2008;358(21):2231-2239. dystrophy. A more severe retinal dystrophy such as LCA 3. Maguire AM, Simonelli F, Pierce EA, et al. Safety and efficacy of gene transfer may result from an excess of null alleles when compared for Leber’s congenital amaurosis. N Engl J Med. 2008;358(21):2240-2248. with other less severe dystrophies such as retinitis pig- 4. Mohamed MD, Topping NC, Jafri H, Raashed Y, McKibbin MA, Inglehearn CF. Progression of phenotype in Leber’s congenital amaurosis with a mutation at mentosa in which visual function may be normal ini- the LCA5 locus. Br J Ophthalmol. 2003;87(4):473-475. tially.14,23 In this series, the family with CRB1-related dis- 5. den Hollander AI, Koenekoop RK, Mohamed MD, et al. Mutations in LCA5, en- ease and 2 of the families with RPGRIP1-related disease coding the ciliary protein lebercilin, cause Leber congenital amaurosis. Nat Genet. 2007;39(7):889-895. were homozygous for nonsense mutations. Four other 6. Stone EM. Leber congenital amaurosis—a model for efficient genetic testing of families had missense mutations, 2 in RPGRIP1 and1in heterogeneous disorders: LXIV Edward Jackson Memorial Lecture. Am J each of TULP1 and RPE65. Based on the available histo- Ophthalmol. 2007;144(6):791-811. logic results from 13 probable LCA cases, LCA may be 7. Damji KF, Sohocki MM, Khan R, et al. Leber’s congenital amaurosis with ante- rior keratoconus in Pakistani families is caused by the Trp278X mutation in the the clinical appearance of either an aplasia in which there AIPL1 gene on 17p. Can J Ophthalmol. 2001;36(5):252-259. is abnormal formation of key elements within the retina, 8. Dryja TP, Adams SM, Grimsby JL, et al. Null RPGRIP1 alleles in patients with a degeneration in which the retina develops correctly but Leber congenital amaurosis. Am J Hum Genet. 2001;68(5):1295-1298. 9. Dharmaraj S, Leroy BP, Sohocki MM, et al. The phenotype of Leber congenital there is early and progressive photoreceptor death, or a amaurosis in patients with AIPL1 mutations. Arch Ophthalmol. 2004;122(7): dysfunction in which the retina is structurally normal ini- 1029-1037. tially but a key biochemical message is missing.1 This dys- 10. Hanein S, Perrault I, Gerber S, et al. Leber congenital amaurosis: comprehen- function may be followed by a secondary degeneration. sive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis. Hum Mutat. However, only 1 of these cases, with RPE65-related dis- 2004;23(4):306-317. ease, was genotyped. For the genes in this series, it seems 11. Jacobson SG, Cideciyan AV, Aleman TS, et al. Leber congenital amaurosis caused possible that mutations in CRB1 might cause an aplasia, by an RPGRIP1 mutation shows treatment potential. Ophthalmology. 2007; given the role in photoreceptor development and struc- 114(5):895-898. 12. Simonelli F, Ziviello C, Testa F, et al. Clinical and molecular genetics of Leber’s ture, and that mutations in all the other genes might cause congenital amaurosis: a multicenter study of Italian patients. Invest Ophthalmol a dysfunction with secondary retinal degeneration, given Vis Sci. 2007;48(9):4284-4290. the various roles in vitamin A metabolism (RPE65), trans- 13. Galvin JA, Fishman GA, Stone EM, Koenekoop RK. Evaluation of genotype- phenotype associations in Leber congenital amaurosis. Retina. 2005;25(7): duction (AIPL1), and intraphotoreceptor ciliary trans- 919-929. 1 port (TULP1, RPGRIP1, and LCA5). However, all 3 reti- 14. Gerber S, Hanein S, Perrault I, et al. Mutations in LCA5 are an uncommon cause nal signs in this study were common but not universal of Leber congenital amaurosis (LCA) type II. Hum Mutat. 2007;28(12):1245. for CRB1-related disease and the frequency of each sign 15. Ramprasad VL, Soumittra N, Nancarrow D, et al. Identification of a novel splice- site mutation in the Lebercilin (LCA5) gene causing Leber congenital amaurosis. was similar to the cases with TULP1- and LCA5-related Mol Vis. 2008;14:481-486. disease. Many retinal signs also became more prevalent 16. Lotery AJ, Jacobson SG, Fishman GA, et al. Mutations in the CRB1 gene cause with increasing age, suggesting that a primary or sec- Leber congenital amaurosis. Arch Ophthalmol. 2001;119(3):415-420. 17. Yzer S, Fishman GA, Racine J, et al. CRB1 heterozygotes with regional retinal ondary degenerative process is common to all LCA genes. dysfunction: implications for genetic testing of Leber congenital amaurosis. In- This concept is supported by the evidence that micro- vest Ophthalmol Vis Sci. 2006;47(9):3736-3744. scopic features precede macroscopic changes.24 18. Yzer S, Leroy BP, De Baere E, et al. Microarray-based mutation detection and In this LCA series from northern Pakistan, muta- phenotypic characterization of patients with Leber congenital amaurosis. Invest Ophthalmol Vis Sci. 2006;47(3):1167-1176. tions were identified in 6 different genes and the disease 19. Thompson DA, Gyürüs P, Fleischer LL, et al. Genetics and phenotypes of RPE65 in another family was linked to the LCA9 locus. The genes mutations in inherited retinal degeneration. Invest Ophthalmol Vis Sci. 2000; most commonly involved in this population differ from 41(13):4293-4299. other series published to date. Visual acuity and signs in 20. Lorenz B, Gyürüs P, Preising M, et al. Early-onset severe rod-cone dystrophy in young children with RPE65 mutations. Invest Ophthalmol Vis Sci. 2000;41 the anterior and posterior segments may be helpful in se- (9):2735-2742. lecting the genes or loci most likely to be implicated in 21. Lotery AJ, Namperumalsamy P, Jacobson SG, et al. Mutation analysis of 3 genes

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 in patients with Leber congenital amaurosis. Arch Ophthalmol. 2000;118(4): 23. Hameed A, Abid A, Aziz A, Ismail M, Mehdi SQ, Khaliq S. Evidence of RPGRIP1 538-543. gene mutations associated with recessive cone-rod dystrophy. J Med Genet. 2003; 22. Fazzi E, Signorini SG, Uggetti C, Bianchi PE, Lanners J, Lanzi G. Towards im- 40(8):616-619. proved clinical characterization of Leber congenital amaurosis: neurological and 24. Porto FB, Perrault I, Hicks D, et al. Prenatal human ocular degeneration occurs systemic findings. Am J Med Genet A. 2005;132A(1):13-19. in Leber’s congenital amaurosis (LCA2). J Gene Med. 2002;4(4):390-396.

Archives Web Quiz Winner

ongratulations to the winner of our August quiz, Veeral S. Sheth, MD, Vitreoretinal Fellow, Section of Ophthal- C mology and Visual Science, Department of Surgery, University of Chicago, Chicago, Illinois. The correct answer to our August challenge was age-related hyperplasia of the nonpigmented ciliary body epithelium (Fuchs adenoma). For a complete discussion of this case, see the Letters: Research Letters section in the September Archives (Shields JA, Shields CL, Eagle RC Jr, Friedman ES, Wheatley HM. Age-related hyperplasia of the nonpigmented ciliary body epithelium [Fuchs adenoma] simulating a ciliary body malignant neoplasm. Arch Ophthalmol. 2009;127[9]:1224). Be sure to visit the Archives of Ophthalmology Web site (http://www.archophthalmol.com) and try your hand at our Clinical Challenge Interactive Quiz. We invite visitors to make a diagnosis based on selected information from a case report or other feature scheduled to be published in the following month’s print edition of the Archives. The first visitor to e-mail our Web editors with the correct answer will be recognized in the print journal and on our Web site and will also be able to choose one of the following books published by AMA Press: Clinical Eye Atlas, Clinical Retina,orUsers’ Guides to the Medical Literature.

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