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

Ramprasad VL, Soumittra N, Nancarrow D, Sen P, McKibbin M, Williams GA, Arokiasamy T, Lakshmipathy P, Inglehearn CF, Kumaramanickavel G - Mol. Vis. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Molecular Biology, Vision Research Foundation, Sankara Nethralaya Chennai, India.

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.

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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.
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f3: 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.

Mentions: In addition, a G>A variation was identified (c.955G>A) in the last base of exon 6, which would be expected to cause a p.Ala319Thr missense mutation. This mutation was homozygous in the affected siblings (Figure 3A) while both parents were heterozygous carriers (Figure 3B). This change was not seen in 50 normal controls of a similar ethnic background. As it affected the last base of exon 6, we used a splice site prediction algorithm [8,9] to investigate the possibility this mutation might also lead to aberrant mRNA splicing. The algorithm calculates scores for potential donor and acceptor sequences that provide an estimate of the strength of these sequences as sites for initiation of splicing. Our analysis of the normal sequence adjacent to the 3' end of exon 6 predicted two donor sites that turned out to be the wild-type exon 6 donor-site and a second donor-site 5 bp into the downstream intron. The algorithm scored these sequences equally as potential donor sites (76.3 and 77.7% respectively). However, when the mutated sequence was tested, the original donor site was no longer predicted. This analysis suggested that the mutation might cause exon 6 to be spliced at the alternative donor site, producing an mRNA with a 5 bp insertion of the intron, breaking the reading frame and potentially truncating the protein (Figure 4).


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

Ramprasad VL, Soumittra N, Nancarrow D, Sen P, McKibbin M, Williams GA, Arokiasamy T, Lakshmipathy P, Inglehearn CF, Kumaramanickavel G - Mol. Vis. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268850&req=5

f3: 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.
Mentions: In addition, a G>A variation was identified (c.955G>A) in the last base of exon 6, which would be expected to cause a p.Ala319Thr missense mutation. This mutation was homozygous in the affected siblings (Figure 3A) while both parents were heterozygous carriers (Figure 3B). This change was not seen in 50 normal controls of a similar ethnic background. As it affected the last base of exon 6, we used a splice site prediction algorithm [8,9] to investigate the possibility this mutation might also lead to aberrant mRNA splicing. The algorithm calculates scores for potential donor and acceptor sequences that provide an estimate of the strength of these sequences as sites for initiation of splicing. Our analysis of the normal sequence adjacent to the 3' end of exon 6 predicted two donor sites that turned out to be the wild-type exon 6 donor-site and a second donor-site 5 bp into the downstream intron. The algorithm scored these sequences equally as potential donor sites (76.3 and 77.7% respectively). However, when the mutated sequence was tested, the original donor site was no longer predicted. This analysis suggested that the mutation might cause exon 6 to be spliced at the alternative donor site, producing an mRNA with a 5 bp insertion of the intron, breaking the reading frame and potentially truncating the protein (Figure 4).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Molecular Biology, Vision Research Foundation, Sankara Nethralaya Chennai, India.

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.

Show MeSH
Related in: MedlinePlus