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Genetic analysis of chromosome 20-related posterior polymorphous corneal dystrophy: genetic heterogeneity and exclusion of three candidate genes.

Hosseini SM, Herd S, Vincent AL, Héon E - Mol. Vis. (2008)

Bottom Line: The three strongest candidate genes of the PPCD1-CHED1 overlap region (RBBP9, ZNF133, SLC24A3) did not show any mutations in our PPCD1-linked families.Mutation analysis of the PPCD1-linked families did not reveal any mutations in the full genomic sequence of VSX1 (considering all splice variants) or in the six cis- regulatory modules predicted in the vicinity of VSX1 (100 kb).This is the first documentation of VSX1 expression in human neonatal cornea.

View Article: PubMed Central - PubMed

Affiliation: Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

ABSTRACT

Purpose: Posterior polymorphous corneal dystrophy (PPCD) is a genetically heterogeneous autosomal dominant condition which maps to the pericentromeric region of chromosome 20. Mutations in the VSX1 transcription factor have been reported in patients affected with PPCD, keratoconus, or a combination of both phenotypes. However, no mutation was identified in the coding region of VSX1 in the family used for the original mapping. To clarify the genetic basis of PPCD1, a thorough analysis was performed on the original PPCD1 family and two other PPCD1-linked families. As part of the analysis, the expression profile, transcript variants, and evolutionary conserved regions of VSX1, a key candidate gene within the linkage interval, were characterized.

Methods: Haplotype analysis was performed using highly informative markers on the pericentromeric region of chromosome 20. VSX1 transcript variants were identified using RT-PCR and characterized by 3'RACE assay. Temporal expression profile of VSX1 was evaluated using semi-quantitative real-time RT-PCR on human tissues. Evolutionary conserved regions (ECRs) were identified in the vicinity of VSX1 using publicly available sequence alignments (UCSC and rVista) and sequenced for mutation analysis.

Results: Recombination events were identified that narrow the PPCD1-disease interval from 20 to 16.44 cM. This smaller interval includes the CHED1 locus and a recently described PPCD locus in Czech families. The three strongest candidate genes of the PPCD1-CHED1 overlap region (RBBP9, ZNF133, SLC24A3) did not show any mutations in our PPCD1-linked families. Semi-quantitative real-time RT-PCR detected VSX1 expression in neonatal human cornea. Six transcript variants of VSX1 were characterized. Four of the transcript variants spliced to two novel exons downstream of the gene. Mutation analysis of the PPCD1-linked families did not reveal any mutations in the full genomic sequence of VSX1 (considering all splice variants) or in the six cis- regulatory modules predicted in the vicinity of VSX1 (100 kb).

Conclusions: This is the first documentation of VSX1 expression in human neonatal cornea. We provide evidence for genetic heterogeneity of chromosome 20-related PPCD and refinement of the original PPCD1 interval. The full genomic sequence of VSX1 and coding exons of three other candidate genes were excluded from being pathogenic in the original PPCD1 family.

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Photograph of ethidium bromide stained 3′RACE products electrophoresed on a 2% sieving agarose gel. Numbers on the left show the sizes of molecular marker (M) bands in bp. Numbers on the right correspond to different VSX1 transcript variants.
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f2: Photograph of ethidium bromide stained 3′RACE products electrophoresed on a 2% sieving agarose gel. Numbers on the left show the sizes of molecular marker (M) bands in bp. Numbers on the right correspond to different VSX1 transcript variants.

Mentions: To identify different mRNA classes transcribed from the VSX1 gene, a 3′RACE experiment was performed on human adult retinal total RNA. Nested PCR using gene specific primers (Table 1, 3OA and 3IA) consistently yielded six polyadenylated products on gel electrophoresis (Figure 2). Duplication of the experiment using another set of primers (Table 1, 3OB and 3IB) produced similar results (data not shown). Sequence analysis of the 3′RACE product clones consistently revealed two novel downstream exons for VSX1, exon 6 and exon 7. Exon 6, 108 bp in length, is located at position 24993279–24993172 on genomic contig NT_011387. Exon 7 spans 535 bp of genomic DNA at position 24992119–24992654 (NT_011387). These two exons were mainly non-coding, not highly conserved and alternatively spliced in different VSX1 transcripts. Including these novel exons, the VSX1 gene now spans 10.65 kb of genomic DNA (former genomic size 6.7 kb).


Genetic analysis of chromosome 20-related posterior polymorphous corneal dystrophy: genetic heterogeneity and exclusion of three candidate genes.

Hosseini SM, Herd S, Vincent AL, Héon E - Mol. Vis. (2008)

Photograph of ethidium bromide stained 3′RACE products electrophoresed on a 2% sieving agarose gel. Numbers on the left show the sizes of molecular marker (M) bands in bp. Numbers on the right correspond to different VSX1 transcript variants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267740&req=5

f2: Photograph of ethidium bromide stained 3′RACE products electrophoresed on a 2% sieving agarose gel. Numbers on the left show the sizes of molecular marker (M) bands in bp. Numbers on the right correspond to different VSX1 transcript variants.
Mentions: To identify different mRNA classes transcribed from the VSX1 gene, a 3′RACE experiment was performed on human adult retinal total RNA. Nested PCR using gene specific primers (Table 1, 3OA and 3IA) consistently yielded six polyadenylated products on gel electrophoresis (Figure 2). Duplication of the experiment using another set of primers (Table 1, 3OB and 3IB) produced similar results (data not shown). Sequence analysis of the 3′RACE product clones consistently revealed two novel downstream exons for VSX1, exon 6 and exon 7. Exon 6, 108 bp in length, is located at position 24993279–24993172 on genomic contig NT_011387. Exon 7 spans 535 bp of genomic DNA at position 24992119–24992654 (NT_011387). These two exons were mainly non-coding, not highly conserved and alternatively spliced in different VSX1 transcripts. Including these novel exons, the VSX1 gene now spans 10.65 kb of genomic DNA (former genomic size 6.7 kb).

Bottom Line: The three strongest candidate genes of the PPCD1-CHED1 overlap region (RBBP9, ZNF133, SLC24A3) did not show any mutations in our PPCD1-linked families.Mutation analysis of the PPCD1-linked families did not reveal any mutations in the full genomic sequence of VSX1 (considering all splice variants) or in the six cis- regulatory modules predicted in the vicinity of VSX1 (100 kb).This is the first documentation of VSX1 expression in human neonatal cornea.

View Article: PubMed Central - PubMed

Affiliation: Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

ABSTRACT

Purpose: Posterior polymorphous corneal dystrophy (PPCD) is a genetically heterogeneous autosomal dominant condition which maps to the pericentromeric region of chromosome 20. Mutations in the VSX1 transcription factor have been reported in patients affected with PPCD, keratoconus, or a combination of both phenotypes. However, no mutation was identified in the coding region of VSX1 in the family used for the original mapping. To clarify the genetic basis of PPCD1, a thorough analysis was performed on the original PPCD1 family and two other PPCD1-linked families. As part of the analysis, the expression profile, transcript variants, and evolutionary conserved regions of VSX1, a key candidate gene within the linkage interval, were characterized.

Methods: Haplotype analysis was performed using highly informative markers on the pericentromeric region of chromosome 20. VSX1 transcript variants were identified using RT-PCR and characterized by 3'RACE assay. Temporal expression profile of VSX1 was evaluated using semi-quantitative real-time RT-PCR on human tissues. Evolutionary conserved regions (ECRs) were identified in the vicinity of VSX1 using publicly available sequence alignments (UCSC and rVista) and sequenced for mutation analysis.

Results: Recombination events were identified that narrow the PPCD1-disease interval from 20 to 16.44 cM. This smaller interval includes the CHED1 locus and a recently described PPCD locus in Czech families. The three strongest candidate genes of the PPCD1-CHED1 overlap region (RBBP9, ZNF133, SLC24A3) did not show any mutations in our PPCD1-linked families. Semi-quantitative real-time RT-PCR detected VSX1 expression in neonatal human cornea. Six transcript variants of VSX1 were characterized. Four of the transcript variants spliced to two novel exons downstream of the gene. Mutation analysis of the PPCD1-linked families did not reveal any mutations in the full genomic sequence of VSX1 (considering all splice variants) or in the six cis- regulatory modules predicted in the vicinity of VSX1 (100 kb).

Conclusions: This is the first documentation of VSX1 expression in human neonatal cornea. We provide evidence for genetic heterogeneity of chromosome 20-related PPCD and refinement of the original PPCD1 interval. The full genomic sequence of VSX1 and coding exons of three other candidate genes were excluded from being pathogenic in the original PPCD1 family.

Show MeSH
Related in: MedlinePlus