The ABCA4 2588G>C Stargardt mutation: single origin and increasing frequency from South-West to North-East Europe.

Inherited retinal dystrophies represent the most important cause of vision impairment in adolescence, affecting approximately 1 out of 3000 individuals. Mutations of the photoreceptor-specific gene ABCA4 (ABCR) are a common cause of retinal dystrophy. A number of mutations have been repeatedly reported for this gene, notably the 2588G>C mutation which is frequent in both patients and controls. Here we ascertained the frequency of the 2588G>C mutation in a total of 2343 unrelated random control individuals from 11 European countries and 241 control individuals from the US, as well as in 614 patients with STGD both from Europe and the US. We found an overall carrier frequency of 1 out of 54 in Europe, compared with 1 out of 121 in the US, confirming that the 2588G>C ABCA4 mutation is one of the most frequent autosomal recessive mutations in the European population. Carrier frequencies show an increasing gradient in Europe from South-West to North-East. The lowest carrier frequency, 0 out of 199 (0%), was found in Portugal; the highest, 11 out of 197 (5.5%), was found in Sweden. Haplotype analysis in 16 families segregating the 2588G>C mutation showed four intragenic polymorphisms invariably present in all 16 disease chromosomes and sharing of the same allele for several markers flanking the ABCA4 locus in most of the disease chromosomes. These results indicate a single origin of the 2588G>C mutation which, to our best estimate, occurred between 2400 and 3000 years ago.

A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line.

BACKGROUND: The rationale of using bovine papillomavirus-1 (BPV-1) derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR) gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. RESULTS: The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. CONCLUSION: Bovine papillomavirus type-1 (BPV-1)-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1-5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

Macular pigment and visual acuity in Stargardt macular dystrophy.

PURPOSE: To test the hypothesis that macular pigment reflects foveal cone function and possibly the presence of foveal cones in recessive Stargardt macular dystrophy. METHODS: Sixteen patients (32 eyes) diagnosed to have Stargardt macular dystrophy by clinical criteria were studied with a scanning laser ophthalmoscope (SLO) comparing argon laser blue (488 nm), green (514), helium-neon laser red (633 nm) and infrared diode laser (780 nm) images for the presence or absence of macular pigment in the fovea. Fifteen of the patients were screened for mutations in the ABCR gene. Eyes were graded into three categories: those without foveal macular pigment, those with partial pigment and those with normal amounts of macular pigment. These categories were compared with visual acuity determined by the Snellen chart. RESULTS: All patients with a visual acuity of 20/200 or worse had no macular pigment in the fovea. All patients with visual acuity of 20/40 or better had a normal amount of macular pigment in the fovea. Patients with partial macular pigment had intermediary acuity values except for two eyes, one with 20/20 and another with 20/200 acuity. Infrared light revealed more retinal abnormalities than blue light at early stages of the disease. CONCLUSION: Foveal macular pigment is related to foveal cone acuity in Stargardt macular dystrophy and may be a marker for the presence of foveal cones. Infrared light is a sensitive monitor of early Stargardt macular dystrophy.

Nonsense mediated decay downregulates conserved alternatively spliced ABCC4 transcripts bearing nonsense codons.

Drug transporters are an important part of the defense of cells against cytotoxic agents. One major group of transporters is known as multidrug resistance associated proteins (MRP; ABCC gene family). The MRPs belong to the ATP binding cassette transporter superfamily. One family member, ABCC4 (also known as MRP4) functions as a cellular efflux pump for anti-HIV drugs, such as 9-(2-phoshoenylmethoxyethyl) adenine and azido-thymidine-monophosphate, an antiviral nucleotide, ganciclovir-monophosphate, and anti-cancer agents such as thiopurines. We isolated a ABCC4 cDNA encoding a non-functional protein, owing to an insertion, and subsequently determined the ABCC4 gene structure. This analysis revealed that the insertion was attributed to two additional exons that would be predicted to produce premature termination codons (PTC) in ABCC4. The highly similar mouse Abcc4 gene also contained these exons, which were remarkable because their size and sequence identity were much higher than the overall similarity between these genes. Further, a comparison of human, monkey and rodent ABCC4 genes revealed that these same PTC-producing exons were also highly conserved in evolution. As all the ABCC4 mRNA containing these PTC exons might produce nonsense mRNA, we further tested the hypothesis that these mRNAs were targets of nonsense-mediated mRNA decay (NMD). Protein synthesis inhibition selectively stabilized PTC containing ABCC4 transcripts in human, monkey and rodent cell lines. Moreover, the amount of PTC-containing ABCC4 transcripts was critically dependent upon protein synthesis, as removal of the inhibitor dramatically decreased expression, which correlated with the resumption of protein synthesis. These are the first studies to indicate that the highly conserved PTC exons of the ABCC4 gene may dictate its expression.