Identification of a novel splice-site mutation in the Lebercilin (LCA5) gene causing Leber congenital amaurosis

Abstract

Purpose: Leber congenital amaurosis (LCA) is one of the most common causes of hereditary blindness in infants. To date, mutations in 13 known genes and at two other loci have been implicated in LCA causation. An examination of the known genes highlights several processes which, when defective, cause LCA, including photoreceptor development and maintenance, phototransduction, vitamin A metabolism, and protein trafficking. In addition, it has been known for some time that defects in sensory cilia can cause syndromes involving hereditary blindness. More recently evidence has come to light that non-syndromic LCA can also be a "ciliopathy."

Methods: Here we present a homozygosity mapping analysis in a consanguineous sibship that led to the identification of a mutation in the recently discovered LCA5 gene. Homozygosity mapping was done using Affymetrix 10K Xba I Gene Chip and a 24.5cM region on chromosome 6 (6q12- q16.3) was identified to be significantly homozygous. The LCA5 gene on this region was sequenced and cDNA sequencing also done to characterize the mutation.

Results: A c.955G>A missense mutation in the last base of exon 6 causing disruption of the splice donor site was identified in both the affected sibs. Since there is a second consensus splice donor sequence 5 bp into the adjacent intron, this mutation results in a transcript with a 5 bp insertion of intronic sequence, leading to a frameshift and premature truncation.

Conclusions: We report a missense mutation functionally altering the splice donor site and leading to a truncated protein. This is the second report of LCA5 mutations causing LCA. It may also be significant that one affected child died at eleven months of age due to asphyxia during sleep. To date the only phenotype unambiguously associated with mutations in this gene is LCA. However the LCA5 gene is known to be expressed in nasopharynx, trachea and lungs and was originally identified in the proteome of bronchial epithelium ciliary axonemes. The cause of death in this child may therefore imply that LCA5 mutations can in fact cause a wider spectrum of phenotypes including respiratory disease.

Figure 1

Color fundus photograph of the right eye. Fundus photograph of the right eye of the proband showing midperipheral white dots at the level of the retinal pigment epithelium, arteriolar attenuation and an abnormal sheen in the macula.

Figure 2

Full field electroretinogram in patient and parents. Full field electroretinogram (ERG) of the proband (A), unaffected father (B), and unaffected mother (C). The ERG is normal for the parents but severely attenuated for the proband.

Figure 3

Mutation analysis of LCA5 gene. A: Sequence chromatogram of the LCA5 gene showing c.955G>A homozygous mutation in the genomic DNA of the affected patient. The homozygous mutation is indicated by the arrow. B: Sequence chromatogram of the LCA5 gene showing c.955G>A heterozygous change in the genomic DNA of the unaffected father. The heterozygous variation is indicated by the arrow. C: cDNA sequence of the LCA5 gene of the affected proband with the mutated splice site. The black square box and the arrow indicate the mutated base. The green dashed box indicates the 5 base insertion of the adjacent intron due to the donor splice site mutation. D: cDNA sequence of a normal control showing the wild type base as indicated by the arrow and the underlined sequence annotation.

Figure 4

Schematic representation of the 3' end of the exon 6 of the LCA5 gene. The figure represents 3' portion of the normal and mutant sequence of the exon 6 of the LCA5 gene. Nucleotides in uppercase represent exonic sequence and that in small lower case represent intronic sequences. The nucleotide in uppercase and in bold represent the last base of the exon and the site of mutation. The first vertical bar in the normal sequence represent the real splice donor site and the second vertical bar represent additional/alternative splice donor site, which is activated in the event of absence of the real splice donor site.