PMC2665199_01
Typical retinal phenotype in affected members A: Left fundus of proband showing temporal optic nerve pallor, attenuated retinal blood vessels, macular pigmentary and atrophic changes, and some fine pigment clumping outside the vascular arcade. B: Mid-transit fluorescein angiogram of left fundus of younger sister reveals a dark choroid sign with some retinal pigment epithelium (RPE) transmission defects in a perifoveal distribution. C and D: Right and left fundus photographs of younger sister at age 12 years show mottling of RPE in macular region but no flagrant pigmentary changes; there is slight temporal pallor of the optic nerve heads, especially in left eye.
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The proband's vision continued to deteriorate slowly, and she developed temporal pallor of her optic nerve head as well as peripheral pigmentary changes in a bony spicule pattern (Figure 1A). There was a dark choroid sign on angiography (Figure 1B). Her macular areas were atrophic (Figure 1C,D).
Linkage of the Stargardt family to ABCA4. The pedigree structure of a family with Stargardt disease and results from linkage analysis and fine mapping to the ABCA4 locus are shown. Circles denote females, and squares indicate males. Dark symbols represent affected individuals with Stargardt disease. Empty symbols are normal individuals. The proband is marked by an arrow. The gray symbol indicates occurrence of Goldenhar syndrome but not Stargardt disease. Genotyping results for markers D1S1588, D1S1170, D1S406, D1S1174, D1S1627, D1S2651, and D1S2726 are shown under each symbol. Haplotypes were constructed on the basis of the minimum number of recombinations between markers. The disease haplotype shared by all affected individuals is denoted by the blackened vertical bar, and normal haplotype is denoted by an empty vertical bar. The pedigree information is as in this figure. The proband is marked by an arrow.
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The inheritance pattern in the family appeared to be autosomal recessive (Figure 2). As shown in Figure 2, haplotype analysis showed that the two affected individuals (II-1 and II-4) carried haplotype 2-1-2-2-1-1-2 from the father and haplotype 1-1-3-1-3-2-3 from the mother.
Identification of two novel ABCA4 mutations. DNA sequence analysis for patient II-1 showed the presence of compound heterozygous c.655A>T and c.5312+3A>T mutations. A and D show the sequences from a normal and affected family member with mutation c.655A>T allele, respectively. B and E show the sequences from a normal and affected family member with mutation c.5312+3A>T allele, respectively. C and F show sequences for the wild type and mutant c.5312+3A>T allele, respectively, which were separated by subcloning of PCR products from an affected family member.
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Two novel mutations were identified (Figure 3). Figure 3A for wild type and Figure 3D for mutant sequence). The other mutation of A to T change (c.5312+3A>T) was identified at a 5' splicing site in intron 37 (Figure 3B for wild type and Figure 3E for mutant sequence). For confirmation of the splicing mutation, PCR products were cloned and multiple clones were sequenced. Mutation c.5312+3A>T was confirmed (Figure 3C showing wild type and Figure 3F showing mutant, both from cloned PCR products).
Cosegregation of two novel ABCA4 mutations with the disease in the family. Sequence analysis on mutations c.655A>T and c.5312+3A>T were done on every family member. Both mutations cosegregated with the disease within the family. Both affected sisters inherited the c.655A>T mutant allele from their father and the c.5312+3A>T mutant allele from their mother. Normal siblings either carried both widetype alleles (II-2, 6, 8, 9) or only one mutant allele (II-3, 5, 7, 10).
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As shown in Figure 4, only the two affected siblings carried both mutations, c.655A>T from the father and c.5312+3A>T from the mother.