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Genotyping and CA4 gene analysis in a Chinese family with retinitis pigmentosa
Abstract
To illuminate pathogenic gene and mutation in a Chinese family with autosomal dominant retinitis pigmentosa (adRP).
Genetic linkage analysis was performed on the known genetic loci for adRP with a panel of polymorphic markers, and then all exons including exon-intron boundary, 5oUTR and 3oUTR of the candidate gene were sequenced directly.
Two-point LOD scores were negative with all markers tested except D17S701 (Zmax=2.107, theta=0) and D17S1604 (Zmax=1.806, theta=0). The disease gene locus was confined to RP17 with further genetic linkage and haplotype analysis. Screening all exons including exon-intron boundary, 5oUTR and 3oUTR of carbonic anhydrase 4 (CA4) revealed no mutation in this family.
The disease-causing gene of one Chinese family with adRP was first mapped to RP17, however no gene mutation of CA4 was detected in this family. Maybe there is a complex CA4 gene mutation in this family or a new disease-causing gene for this family in this locus, further study need to be done.
To investigate the significance of carbonic anhydrase IV gene mutations in sporadic retinitis pigmentosa (RP) among the Chinese population.
A total of 116 patients with simplex RP and adRP of Chinese ethnicity were screened for mutations in the eight coding exons of the CA4 gene by sequencing. Immunostaining and Real-Time PCR were used to measure the cellular localization and expression level of CA4 variations.
Ten sequence alterations were identified, including a novel variant within exon 1 of CA4 (A12T) in a patient with RP. No notable difference was found between wild-type protein and A12T mutant protein in the distribution pattern. However, there was a decrease in level of mRNA expression of 3' UTR mutant to wild-type.
The results suggest that the expression level of CA IV may be important to maintain retina function in RP.
Retinitis pigmentosa is one of the most common causes of severe visual handicap in middle to late life. Prior to this report,
seven loci had previously been mapped for the autosomal dominant form of this disorder (adRP). We now report the identification
of a novel adRP locus on chromosome 17q. To map the new locus, we performed linkage analysis with microsatellite markers in
a large South African kindred. After exclusion of 13 RP candidate gene loci (including rhodopsin and peripherin-RDS), we obtained
significant positive lod scores at zero recombination fraction (θ = 0) for D17S808 (Z = 4.63) and D17S807 (Z = 5.69). Multipoint
analysis gave a maximum lod score of 8.28 between these two markers. From haplotype analysis, the disease locus lies in the
interval between markers D17S809 and D17S942. Three candidate genes for retinal dystrophies map to this chromosomal region
and these genes are currently being investigated for possible involvement with adRP in this family.
Carbonic anhydrase IV (CA IV) is a glycosylphosphatidylinositol-anchored membrane isozyme expressed on the luminal surfaces of pulmonary (and certain other) capillaries and on the luminal surface of proximal renal tubules. It is of interest for its functional importance in CO2 and HCO3- transport, its ancient evolutionary status among CA isozymes, and its possible role in inherited renal abnormalities of HCO3- transport. To determine the localization of the CA IV gene and define its genomic structure, we isolated and characterized a full-length genomic clone. The 9.5-kb gene contains eight exons and seven introns. The first exon (exon 1a) encodes the signal sequence. Exons 1b through 7 encode the remaining coding sequence. Exon 7 encodes the C terminus of the enzyme precursor, the C terminus of the mature protein and also contains the 120-bp sequence corresponding to the 3' untranslated region of the cDNA. The positions of introns 3, 4, 5, and 6 are identical with the corresponding positions of introns in the genes for the soluble CA isozymes (CA I, II, III, and VII). However, intron 1a is not found in these genes, and the positions of introns 1b and 2 in CA IV differ from the positions of the corresponding introns in genes for the soluble isozymes. The 5' upstream region of the gene (-500 to -1) is GC rich and contains 30 CpG dinucleotides. A TATA box sequence and a potential Sp1 binding site are identified upstream of the first exon. By using PCR on DNA from human/rodent somatic cell hybrids, the CA IV gene was assigned to chromosome 17.(ABSTRACT TRUNCATED AT 250 WORDS)
This group has previously reported the mapping of a novel locus for autosomal dominant retinitis pigmentosa (adRP) in a South African kindred to 17q. Using a new series of microsatellite markers in this study, twopoint and multipoint analysis provide evidence for the localization of the disease gene to the 17q22 region. In addition, a second South African adRP family is shown to be linked to this 17q22 locus. Disease-associated haplotypes constructed for both families and multipoint linkage analysis place the gene in the 10-cM interval between D17S1607 and D17S1874. Three candidate genes on 17q were investigated: PDEG, the gamma subunit of rod phosphodiesterase; TIMP2, tissue inhibitor of metalloproteinases-2; and PRKCA, protein kinase C alpha. Recombination events between the adRP locus and: (1) a single-stranded conformation polymorphism in PDEG; and (2) a restriction fragment length polymorphism in TIMP2 provided evidence for the exclusion of these candidate genes as being responsible for adRP in the South African kindred.