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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

Causal variants in PDE6A identified in families PKRP133 and PKRP140. A: Sequence chromatogram of unaffected individual 15 homozygous for the wild-type. B: Sequence chromatogram of unaffected individual 28 heterozygous carriers. C: Sequence chromatogram of affected individual 29 of PKRP133 homozygous for the G to A variation in intron 16; c.2028–1G>A. D: Pedigree of family PKRP140 with the haplotypes of four adjacent chromosome 5q33 microsatellite markers are shown with the alleles forming the risk haplotype shaded black and the wild-type allele shown in white. Squares are males, circles are females, filled symbols are affected individuals, the double line between individuals indicates consanguinity, and the diagonal line through a symbol is a deceased family member. E: Sequence chromatogram of unaffected control homozygous for the wild-type. F: Sequence chromatogram of individual 13 heterozygous carrier. G: Sequence chromatogram of affected individual 14 of PKRP140 homozygous for the A to G transition in intron 10: c.1408–2A>G. The arrows point to the splice site variation identified in families PKRP133 (c.2028–1G) and PRKP140 (c.1408–2A) whereas green, red, and brown represent the exon, splice acceptor site, and intron, respectively. SA=splice acceptor.
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f4: Causal variants in PDE6A identified in families PKRP133 and PKRP140. A: Sequence chromatogram of unaffected individual 15 homozygous for the wild-type. B: Sequence chromatogram of unaffected individual 28 heterozygous carriers. C: Sequence chromatogram of affected individual 29 of PKRP133 homozygous for the G to A variation in intron 16; c.2028–1G>A. D: Pedigree of family PKRP140 with the haplotypes of four adjacent chromosome 5q33 microsatellite markers are shown with the alleles forming the risk haplotype shaded black and the wild-type allele shown in white. Squares are males, circles are females, filled symbols are affected individuals, the double line between individuals indicates consanguinity, and the diagonal line through a symbol is a deceased family member. E: Sequence chromatogram of unaffected control homozygous for the wild-type. F: Sequence chromatogram of individual 13 heterozygous carrier. G: Sequence chromatogram of affected individual 14 of PKRP140 homozygous for the A to G transition in intron 10: c.1408–2A>G. The arrows point to the splice site variation identified in families PKRP133 (c.2028–1G) and PRKP140 (c.1408–2A) whereas green, red, and brown represent the exon, splice acceptor site, and intron, respectively. SA=splice acceptor.

Mentions: Subsequently, we genotyped additional STR markers proximal to D5S636, the previously defined proximal boundary of the critical interval. Lack of homozygosity in affected individuals 21, 27, and 29 at marker D5S2090 suggested the causal mutation lies distal to marker D5S2090 extending the critical interval with significant two-point LOD scores (Table 2). The new critical interval included PDE6A, a gene previously associated with RP. We sequenced all the coding exons, exon–intron boundaries along with the 5′ and 3′ untranslated regions (UTRs) of PDE6A that identified a variation in intron 16 at the splice acceptor site substituting guanine for adenosine at position −1: c.2028–1G>A. The mutation segregated with disease phenotype in the family (Figure 1 and Figure 4A-C) except individual 17 (heterozygous carrier of the c.2028–1G>A variation) and was absent in 350 ethnically matched control chromosomes. This variation was not present in the 1000 Genomes and NHLBI Exome Sequencing Project databases.


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)

Causal variants in PDE6A identified in families PKRP133 and PKRP140. A: Sequence chromatogram of unaffected individual 15 homozygous for the wild-type. B: Sequence chromatogram of unaffected individual 28 heterozygous carriers. C: Sequence chromatogram of affected individual 29 of PKRP133 homozygous for the G to A variation in intron 16; c.2028–1G>A. D: Pedigree of family PKRP140 with the haplotypes of four adjacent chromosome 5q33 microsatellite markers are shown with the alleles forming the risk haplotype shaded black and the wild-type allele shown in white. Squares are males, circles are females, filled symbols are affected individuals, the double line between individuals indicates consanguinity, and the diagonal line through a symbol is a deceased family member. E: Sequence chromatogram of unaffected control homozygous for the wild-type. F: Sequence chromatogram of individual 13 heterozygous carrier. G: Sequence chromatogram of affected individual 14 of PKRP140 homozygous for the A to G transition in intron 10: c.1408–2A>G. The arrows point to the splice site variation identified in families PKRP133 (c.2028–1G) and PRKP140 (c.1408–2A) whereas green, red, and brown represent the exon, splice acceptor site, and intron, respectively. SA=splice acceptor.
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f4: Causal variants in PDE6A identified in families PKRP133 and PKRP140. A: Sequence chromatogram of unaffected individual 15 homozygous for the wild-type. B: Sequence chromatogram of unaffected individual 28 heterozygous carriers. C: Sequence chromatogram of affected individual 29 of PKRP133 homozygous for the G to A variation in intron 16; c.2028–1G>A. D: Pedigree of family PKRP140 with the haplotypes of four adjacent chromosome 5q33 microsatellite markers are shown with the alleles forming the risk haplotype shaded black and the wild-type allele shown in white. Squares are males, circles are females, filled symbols are affected individuals, the double line between individuals indicates consanguinity, and the diagonal line through a symbol is a deceased family member. E: Sequence chromatogram of unaffected control homozygous for the wild-type. F: Sequence chromatogram of individual 13 heterozygous carrier. G: Sequence chromatogram of affected individual 14 of PKRP140 homozygous for the A to G transition in intron 10: c.1408–2A>G. The arrows point to the splice site variation identified in families PKRP133 (c.2028–1G) and PRKP140 (c.1408–2A) whereas green, red, and brown represent the exon, splice acceptor site, and intron, respectively. SA=splice acceptor.
Mentions: Subsequently, we genotyped additional STR markers proximal to D5S636, the previously defined proximal boundary of the critical interval. Lack of homozygosity in affected individuals 21, 27, and 29 at marker D5S2090 suggested the causal mutation lies distal to marker D5S2090 extending the critical interval with significant two-point LOD scores (Table 2). The new critical interval included PDE6A, a gene previously associated with RP. We sequenced all the coding exons, exon–intron boundaries along with the 5′ and 3′ untranslated regions (UTRs) of PDE6A that identified a variation in intron 16 at the splice acceptor site substituting guanine for adenosine at position −1: c.2028–1G>A. The mutation segregated with disease phenotype in the family (Figure 1 and Figure 4A-C) except individual 17 (heterozygous carrier of the c.2028–1G>A variation) and was absent in 350 ethnically matched control chromosomes. This variation was not present in the 1000 Genomes and NHLBI Exome Sequencing Project databases.

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