Homozygosity Mapping in Leber Congenital Amaurosis and Autosomal Recessive Retinitis Pigmentosa in South Indian Families

Abstract

Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP) are retinal degenerative diseases which cause severe retinal dystrophy affecting the photoreceptors. LCA is predominantly inherited as an autosomal recessive trait and contributes to 5% of all retinal dystrophies; whereas RP is inherited by all the Mendelian pattern of inheritance and both are leading causes of visual impairment in children and young adults. Homozygosity mapping is an efficient strategy for mapping both known and novel disease loci in recessive conditions, especially in a consanguineous mating, exploiting the fact that the regions adjacent to the disease locus will also be homozygous by descent in such inbred children. Here we have studied eleven consanguineous LCA and one autosomal recessive RP (arRP) south Indian families to know the prevalence of mutations in known genes and also to know the involvement of novel loci, if any. Complete ophthalmic examination was done for all the affected individuals including electroretinogram, fundus photograph, fundus autofluorescence, and optical coherence tomography. Homozygosity mapping using Affymetrix 250K HMA GeneChip on eleven LCA families followed by screening of candidate gene(s) in the homozygous block identified mutations in ten families; AIPL1 - 3 families, RPE65- 2 families, GUCY2D, CRB1, RDH12, IQCB1 and SPATA7 in one family each, respectively. Six of the ten (60%) mutations identified are novel. Homozygosity mapping using Affymetrix 10K HMA GeneChip on the arRP family identified a novel nonsense mutation in MERTK. The mutations segregated within the family and was absent in 200 control chromosomes screened. In one of the eleven LCA families, the causative gene/mutation was not identified but many homozygous blocks were noted indicating that a possible novel locus/gene might be involved. The genotype and phenotype features, especially the fundus changes for AIPL1, RPE65, CRB1, RDH12 genes were as reported earlier.

Fig 1. Segregation analysis.

1a:arRP1 MERTK c.721C>T, 1b:LCA-1 RPE65 c.850+1G>T, 1c: LCA-2 CRB1 c.3307G>A, 1d:LCA-3 GUCY2D c.994delC, 1e:LCA-4 IQCB1 c.1278+6T>A, 1f:LCA-5 AIPL1 c.824G>A, 1g: LCA-7 RDH12 c.344-8C>T, 1h:LCA-8 AIPL1 c.247G>A, 1i:LCA-9 RPE65 c.1409C>T, 1j:LCA-10 AIPL1 c.613_622 delATCATCTGCC, 1k:LCA-11 SPATA7 c.913-2A>G. The arrow indicates the index case. The filled in circles and squares are affected females and males respectively. [M];[M]–affected with homozygous mutation, [M]; [=] –carries for any given mutation and [=]; [=] –wild type. Lines above the individual indicate availability of genotype.

Fig 2. 2% Agarose gel electrophoresis showing cDNA amplification of exon 11–13 of IQCB1.

Lane 1-100bp ladder, Lane 3- Affected index case, Lane 5 & 7—Carrier parents, Lane 9—Control, Lane 2, 4, 6, 8—empty wells Fig 2b Eletrophoretogram trace showing the amplified cDNA of control and proband. In proband exon 11 is followed by exon 13 and exon 12 is completely deleted, whereas in control, exon 11, 12 and 13 is continuous. The end of exon 11 is marked in both the phoretograms.

Fig 3. Fundus photographs.

Fig 3a A 10yrs old female with c.824G>A p.(Trp278*) mutation in AIPL1 (LCA-5 family) showed normal disc, attenuated vessels, (arrow mark indicates) yellow patches in macula. Fig 3b A 14yrs old male with c.824G>A p.(Trp278*) mutation in AIPL1 (LCA-5 family, elder sibling) showed normal disc, attenuated vessels, (arrow mark indicates) black pigments in macula. Fig 3c A 18 yrs old female with c.850+1G>T (r.spl?) mutation in RPE65 (LCA-1 family) showed pallor disc, attenuated vessels with scar in the macula, peripheral RPE mottling (marked with arrow) Fig 3d A 28yrs old male with c.1409C>T p.(Pro470Leu) mutation in RPE65 (LCA-9 family) showed pallor disc, attenuated vessels, normal macula, with salt and pepper fundus. Arrow mark shows distinct pin head size yellow white dot like spots at the posterior pole. Fig 3e A 14 yrs old female with c.2971G>A p.(Gly991Arg) mutation in CRB1 (LCA-2 family) showed coin shaped pigment clumps and greyish atrophic changes seen in the macula, (arrow mark indicates the macula) Fig 3f A 18 yrs old female with c.2971G>A p.(Gly991Arg) mutation in CRB1 (LCA-2 family, elder sibling) showed pale disc, attenuated vessels, atrophic macula with nummular pigment clumps and greyish atrophic reflex (arrow mark indicates the macula) Fig 3g A 19yrs old male female with c.2971G>A p.(Gly991Arg) mutation in CRB1 (LCA-2 family, eldest sibling) showed coin shaped pigment clumps seen in the background (arrow mark indicates the coin shaped clumps) All the three affected siblings show progressive changes in macula with age for CRB1 mutation positive family. Fig 3h, 3i, 3j A 24 yrs old female, a 25 yrs old female and a 32 yrs old female with c.721C>T p.(Gln 241*) mutation in MERTK (arRP1 family) showing mild, milder and marked features of RP, respectively. Progressive changes with age in the macula are observed.