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Splice-site mutations identified in PDE6A responsible for retinitis pigmentosa in consanguineous Pakistani families.

Khan SY, Ali S, Naeem MA, Khan SN, Husnain T, Butt NH, Qazi ZA, Akram J, Riazuddin S, Ayyagari R, Hejtmancik JF, Riazuddin SA - Mol. Vis. (2015)

Bottom Line: An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP.Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family.Taken together with our previously published work, our data suggest that mutations in PDE6A account for about 2% of the total genetic load of RP in our cohort and possibly in the Pakistani population as well.

View Article: PubMed Central - PubMed

Affiliation: The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore MD.

ABSTRACT

Purpose: This study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin.

Methods: Ophthalmic examinations that included funduscopy and electroretinography (ERG) were performed to confirm the affectation status. Blood samples were collected from all participating individuals, and genomic DNA was extracted. A genome-wide scan was performed, and two-point logarithm of odds (LOD) scores were calculated. Sanger sequencing was performed to identify the causative variants. Subsequently, we performed whole exome sequencing to rule out the possibility of a second causal variant within the linkage interval. Sequence conservation was performed with alignment analyses of PDE6A orthologs, and in silico splicing analysis was completed with Human Splicing Finder version 2.4.1.

Results: A large multigenerational consanguineous family diagnosed with early-onset RP was ascertained. An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP. A genome-wide scan was performed, and suggestive two-point LOD scores were observed with markers on chromosome 5q. Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family. Subsequently, we performed a second genome-wide scan that excluded the entire genome except the chromosome 5q region harboring PDE6A. Next-generation whole exome sequencing identified a splice acceptor site mutation in intron 16: c.2028-1G>A, which was completely conserved in PDE6A orthologs and was absent in ethnically matched 350 control chromosomes, the 1000 Genomes database, and the NHLBI Exome Sequencing Project. Subsequently, we investigated our entire cohort of RP familial cases and identified a second family who harbored a splice acceptor site mutation in intron 10: c.1408-2A>G. In silico analysis suggested that these mutations will result in the elimination of wild-type splice acceptor sites that would result in either skipping of the respective exon or the creation of a new cryptic splice acceptor site; both possibilities would result in retinal photoreceptor cells that lack PDE6A wild-type protein.

Conclusions: we report two splice acceptor site variations in PDE6A in consanguineous Pakistani families who manifested cardinal symptoms of RP. Taken together with our previously published work, our data suggest that mutations in PDE6A account for about 2% of the total genetic load of RP in our cohort and possibly in the Pakistani population as well.

No MeSH data available.


Related in: MedlinePlus

Predictive outcome of PDE6A splice acceptor site variations on the exon splicing mechanism. A: Ideograms illustrate the skipping of exon 11 due to a splice acceptor site mutation at position −2 of intron 10: c.1408–2A>G. B–D: Ideograms illustrate the predictive splicing patterns of exon 17 due to the splice acceptor site mutation at position −1 of intron 16: c.2028–1G>A. B: The prediction of a strong consensus value (CV) of 82.23 for the wild-type splice acceptor site (c.2028–1G). C: A possible scenario that would result in the skipping of exon 17 during mRNA processing. D: Predicting the creation of a new cryptic splice acceptor site (CV=74.31) with predicted deviation (∆CV) of +63.81% that would result in the premature stop codon (p.K677Rfs24*) and eventually would lead to a truncated protein. CV=consensus values; ∆CV=reduction in consensus values.
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f6: Predictive outcome of PDE6A splice acceptor site variations on the exon splicing mechanism. A: Ideograms illustrate the skipping of exon 11 due to a splice acceptor site mutation at position −2 of intron 10: c.1408–2A>G. B–D: Ideograms illustrate the predictive splicing patterns of exon 17 due to the splice acceptor site mutation at position −1 of intron 16: c.2028–1G>A. B: The prediction of a strong consensus value (CV) of 82.23 for the wild-type splice acceptor site (c.2028–1G). C: A possible scenario that would result in the skipping of exon 17 during mRNA processing. D: Predicting the creation of a new cryptic splice acceptor site (CV=74.31) with predicted deviation (∆CV) of +63.81% that would result in the premature stop codon (p.K677Rfs24*) and eventually would lead to a truncated protein. CV=consensus values; ∆CV=reduction in consensus values.

Mentions: We used HSF, a freely available online bioinformatics tool, to predict the effect of the c.1408–2A and c.2028–1G variations on PDE6A mRNA splicing. The HSF analysis generated consensus values of 81.30 and 52.35 for the wild-type (c.1408–2A) and mutant (of c.1408–2G) nucleotides, respectively. The predicted consensus deviation value of −35.61% for c.1408–2A>G suggests the loss of the wild-type splice site that would result in the skipping of exon 11 of PDE6A (Figure 6A). Likewise, HSF analysis predicted the consensus values of 82.23 and 53.28 for the wild-type (c.2028–1G) and mutant (c.2028–1A) nucleotides, respectively. The predicted deviation of −35.21% for c.2028–1G>A suggests the loss of the wild-type splice acceptor site of exon 17 (Figure 6B-C). In parallel, HSF also detected the creation of a new cryptic splice acceptor site using the G nucleotide (c.2028G) in exon 17 (Figure 6D). The algorithm predicted consensus values of 45.37 and 74.31 for the wild-type (c.2028–1G) and the new cryptic splice site (c.2028G), respectively. The predicted consensus value deviation of +63.81% for the new cryptic splice acceptor site suggests the loss of the wild-type splice site that would result in a frameshift and eventually would lead to a premature stop codon (p.K677Rfs24*) in the protein.


Splice-site mutations identified in PDE6A responsible for retinitis pigmentosa in consanguineous Pakistani families.

Khan SY, Ali S, Naeem MA, Khan SN, Husnain T, Butt NH, Qazi ZA, Akram J, Riazuddin S, Ayyagari R, Hejtmancik JF, Riazuddin SA - Mol. Vis. (2015)

Predictive outcome of PDE6A splice acceptor site variations on the exon splicing mechanism. A: Ideograms illustrate the skipping of exon 11 due to a splice acceptor site mutation at position −2 of intron 10: c.1408–2A>G. B–D: Ideograms illustrate the predictive splicing patterns of exon 17 due to the splice acceptor site mutation at position −1 of intron 16: c.2028–1G>A. B: The prediction of a strong consensus value (CV) of 82.23 for the wild-type splice acceptor site (c.2028–1G). C: A possible scenario that would result in the skipping of exon 17 during mRNA processing. D: Predicting the creation of a new cryptic splice acceptor site (CV=74.31) with predicted deviation (∆CV) of +63.81% that would result in the premature stop codon (p.K677Rfs24*) and eventually would lead to a truncated protein. CV=consensus values; ∆CV=reduction in consensus values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4539017&req=5

f6: Predictive outcome of PDE6A splice acceptor site variations on the exon splicing mechanism. A: Ideograms illustrate the skipping of exon 11 due to a splice acceptor site mutation at position −2 of intron 10: c.1408–2A>G. B–D: Ideograms illustrate the predictive splicing patterns of exon 17 due to the splice acceptor site mutation at position −1 of intron 16: c.2028–1G>A. B: The prediction of a strong consensus value (CV) of 82.23 for the wild-type splice acceptor site (c.2028–1G). C: A possible scenario that would result in the skipping of exon 17 during mRNA processing. D: Predicting the creation of a new cryptic splice acceptor site (CV=74.31) with predicted deviation (∆CV) of +63.81% that would result in the premature stop codon (p.K677Rfs24*) and eventually would lead to a truncated protein. CV=consensus values; ∆CV=reduction in consensus values.
Mentions: We used HSF, a freely available online bioinformatics tool, to predict the effect of the c.1408–2A and c.2028–1G variations on PDE6A mRNA splicing. The HSF analysis generated consensus values of 81.30 and 52.35 for the wild-type (c.1408–2A) and mutant (of c.1408–2G) nucleotides, respectively. The predicted consensus deviation value of −35.61% for c.1408–2A>G suggests the loss of the wild-type splice site that would result in the skipping of exon 11 of PDE6A (Figure 6A). Likewise, HSF analysis predicted the consensus values of 82.23 and 53.28 for the wild-type (c.2028–1G) and mutant (c.2028–1A) nucleotides, respectively. The predicted deviation of −35.21% for c.2028–1G>A suggests the loss of the wild-type splice acceptor site of exon 17 (Figure 6B-C). In parallel, HSF also detected the creation of a new cryptic splice acceptor site using the G nucleotide (c.2028G) in exon 17 (Figure 6D). The algorithm predicted consensus values of 45.37 and 74.31 for the wild-type (c.2028–1G) and the new cryptic splice site (c.2028G), respectively. The predicted consensus value deviation of +63.81% for the new cryptic splice acceptor site suggests the loss of the wild-type splice site that would result in a frameshift and eventually would lead to a premature stop codon (p.K677Rfs24*) in the protein.

Bottom Line: An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP.Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family.Taken together with our previously published work, our data suggest that mutations in PDE6A account for about 2% of the total genetic load of RP in our cohort and possibly in the Pakistani population as well.

View Article: PubMed Central - PubMed

Affiliation: The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore MD.

ABSTRACT

Purpose: This study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin.

Methods: Ophthalmic examinations that included funduscopy and electroretinography (ERG) were performed to confirm the affectation status. Blood samples were collected from all participating individuals, and genomic DNA was extracted. A genome-wide scan was performed, and two-point logarithm of odds (LOD) scores were calculated. Sanger sequencing was performed to identify the causative variants. Subsequently, we performed whole exome sequencing to rule out the possibility of a second causal variant within the linkage interval. Sequence conservation was performed with alignment analyses of PDE6A orthologs, and in silico splicing analysis was completed with Human Splicing Finder version 2.4.1.

Results: A large multigenerational consanguineous family diagnosed with early-onset RP was ascertained. An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP. A genome-wide scan was performed, and suggestive two-point LOD scores were observed with markers on chromosome 5q. Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family. Subsequently, we performed a second genome-wide scan that excluded the entire genome except the chromosome 5q region harboring PDE6A. Next-generation whole exome sequencing identified a splice acceptor site mutation in intron 16: c.2028-1G>A, which was completely conserved in PDE6A orthologs and was absent in ethnically matched 350 control chromosomes, the 1000 Genomes database, and the NHLBI Exome Sequencing Project. Subsequently, we investigated our entire cohort of RP familial cases and identified a second family who harbored a splice acceptor site mutation in intron 10: c.1408-2A>G. In silico analysis suggested that these mutations will result in the elimination of wild-type splice acceptor sites that would result in either skipping of the respective exon or the creation of a new cryptic splice acceptor site; both possibilities would result in retinal photoreceptor cells that lack PDE6A wild-type protein.

Conclusions: we report two splice acceptor site variations in PDE6A in consanguineous Pakistani families who manifested cardinal symptoms of RP. Taken together with our previously published work, our data suggest that mutations in PDE6A account for about 2% of the total genetic load of RP in our cohort and possibly in the Pakistani population as well.

No MeSH data available.


Related in: MedlinePlus