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. Lwith 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. (REPRINTED) ARCH OPHTHALMOL / VOL 128 (NO. 1), JAN 2010 WWW.ARCHOPHTHALMOL.COM 107 ©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 (REPRINTED) ARCH OPHTHALMOL / VOL 128 (NO. 1), JAN 2010 WWW.ARCHOPHTHALMOL.COM 108 ©2010 American Medical Association.