Five major sequence variants and copy number variants in the EYS gene account for one-third of Japanese patients with autosomal recessive and simplex retinitis pigmentosa.

Purpose: To elucidate the variant spectrum of the EYS gene in a large cohort of Japanese patients with autosomal recessive and simplex retinitis pigmentosa (arRP and sRP). Methods: We performed a direct sequencing analysis of 44 exons of the EYS gene in 469 patients with RP (including 144 arRP, 288 sRP, and 17 autosomal dominant RP (adRP) cases) in eastern and western regions of Japan and a multiplex ligation-dependent probe amplification (MLPA) of patients who had a single heterozygous pathogenic variant. Results: We identified six pathogenic and 16 likely pathogenic variants from a total of 186 nucleotide sequence variants, of which five variants, c.2528G>A (p.(Gly843Glu)), c.4957dupA (p.(Ser1653Lysfs*2)), c.6557G>A (p.(Gly2186Glu)), c.6563T>C (p.(Ile2188Thr)), and c.8868C>A (p.(Tyr2956*)), were prevalent in patients with arRP and sRP. The homozygous and heterozygous combinations of these five variants accounted for 32.4% (140/432) of Japanese patients with arRP and sRP. Five patients with adRP also had these variants. These five variants segregated with the phenotype in 15 families with RP. MLPA revealed seven copy number variations (CNVs) of the EYS exon(s). Conclusions: This study showed that five major sequence variants and CNVs in the EYS gene account for one-third of Japanese patients with arRP and sRP, and these variants are also responsible for RP showing an autosomal dominant inheritance pattern. This is the first report showing the pathogenicity of three missense variants (p.(Gly843Glu), p.(Gly2186Glu), and p.(Ile2188Thr)) and the presence of CNVs in the EYS gene of Japanese patients with arRP and sRP.

The manner of decay of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts derived from retinitis pigmentosa patients depends on the type of mutation.

BACKGROUND: Generation of induced photoreceptors holds promise for in vitro modeling of intractable retinal diseases. Retinitis pigmentosa is an inherited retinal dystrophy that leads to visual impairment. The EYS gene was reported to be the most common gene responsible for autosomal recessive retinitis pigmentosa (arRP). arRP with defects in the EYS gene is denoted by "EYS-RP". We previously established a "redirect differentiation" method to generate photosensitive photoreceptor-like cells from commercially available human dermal fibroblasts. In this study, we produced photoreceptor-like cells from dermal fibroblasts of EYS-RP patients as a replacement for the degenerative retinas using "redirect differentiation". We analyzed defective transcripts of the EYS gene in these cells to elucidate phenotypes of EYS-RP patients because decay of transcripts was previously suggested to be involved in phenotypic variation associated with diseases. METHODS: Using "redirect differentiation" by CRX, RAX, NeuroD and OTX2, we made photoreceptor-directed fibroblasts derived from three normal volunteers and three EYS-RP patients with homozygous or heterozygous mutations. We tested inducible expression of the photoreceptor-specific genes (blue opsin, rhodopsin, recoverin, S-antigen, PDE6C) in these cells. We then analyzed transcripts derived from three different types of the defective EYS gene, c.1211dupA, c.4957dupA and c.8805C > A, expressed in these cells by RT-PCR and sequencing. RESULTS: Photoreceptor-specific genes including the EYS gene were up-regulated in all the photoreceptor-directed fibroblasts tested. However, expression levels of defective transcripts were markedly different depending on the type of mutation. Transcripts derived from these three defective genes were scarcely detected, expressed at a lower level, and expressed at almost the same level as in normal volunteers, respectively. CONCLUSIONS: Expression levels of genetically defective EYS gene transcripts in photoreceptor-directed fibroblasts of EYS-RP patients vary depending on the type of mutation. Variation in expression levels in transcripts having c.1211dupA, c.4957dupA and c.8805C > A suggests that almost complete nonsense-mediated mRNA decay (NMD), partial NMD and escape from NMD occurred for these transcripts, respectively. To determine the relationship with phenotypic variations in EYS-RP patients, more samples are needed. The present study also suggests that the redirect differentiation method could be a valuable tool for disease modeling despite some limitations.

High prevalence of mutations in the EYS gene in Japanese patients with autosomal recessive retinitis pigmentosa.

PURPOSE: To screen for disease-causing mutations in the Eyes shut homolog (EYS) gene in Japanese patients with retinitis pigmentosa (RP). Methods. Blood samples were obtained from 68 RP patients and 68 controls. Genomic DNA was extracted from the blood samples and used for screening of mutations in the coding exons by direct sequencing. Each patient underwent a detailed clinical examination. RESULTS: Nine nucleotide sequence variations causing amino acid changes were observed in homozygous or heterozygous alleles in 26 patients but not in 68 controls. Seven truncating mutations were found in 21 (32.8%) of 64 patients with nonsyndromic RP composed of 23 autosomal recessive RP (arRP) and 41 sporadic cases. The most abundant mutation was p.S1653Kfs*2, which was generated by a single adenine insertion into exon 26 (c.4957dupA) and was carried by 15 patients. The mutation p.Y2935*, produced by a single nucleotide substitution (c.8805C>A) in the last exon, was carried by five patients. These two truncating mutations were probably founder mutations because each was carried by the particular haplotype. The patients with homozygous or compound heterozygous truncating mutations showed a severe decline in visual acuity, whereas those with a single truncating mutation showed a mild decline. CONCLUSIONS: One-third of Japanese patients with nonsyndromic arRP carried probable pathogenic mutations in the EYS gene, including two founder mutations. Because the genotype was correlated with the phenotype, genotyping in the EYS gene could be a valuable tool for predicting long-term prognoses of Japanese patients with arRP and thus could be useful for genetic counseling and future gene therapy.

Full-length transcriptome analysis of human retina-derived cell lines ARPE-19 and Y79 using the vector-capping method.

PURPOSE. To collect an entire set of full-length cDNA clones derived from human retina-derived cell lines and to identify full-length transcripts for retinal preferentially expressed genes. METHODS. The full-length cDNA libraries were constructed from a retinoblastoma cell line, Y79, and a retinal pigment epithelium cell line, ARPE-19, using the vector-capping method, which generates a genuine full-length cDNA. By single-pass sequencing of the 5'-end of cDNA clones and subsequent mapping to the human genome, the authors determined their transcriptional start sites and annotated the cDNA clones. RESULTS. Of the 23,616 clones isolated from Y79-derived cDNA libraries, 19,229 full-length cDNA clones were identified and classified into 4808 genes, including genes of >10 kbp. Of the 7067 genes obtained from the Y79 and ARPE-19 libraries, the authors selected 72 genes that were preferentially expressed in the eye, of which 131 clones corresponding to 57 genes were fully sequenced. As a result, we discovered many variants that were produced by different transcriptional start sites, alternative splicing, and alternative polyadenylation. CONCLUSIONS. The bias-free, full-length cDNA libraries constructed using the vector-capping method were shown to be useful for collecting an entire set of full-length cDNA clones for these retinal cell lines. Full-length transcriptome analysis of these cDNA libraries revealed that there were, unexpectedly, many transcript variants for each gene, indicating that obtaining the full-length cDNA for each variant is indispensable for analyzing its function. The full-length cDNA clones (approximately 80,000 clones each for ARPE-19 and Y79) will be useful as a resource for investigating the human retina.

Full-length transcriptome analysis using a bias-free cDNA library prepared with the vector-capping method.

Full-length complementary DNAs (cDNAs) are an essential resource for functional genomics. Recently, we have developed a simple and efficient method for preparing a full-length cDNA library from a small amount of total RNA, named the "vector-capping" method. The biggest advantage of this method is that the intactness of the cDNA can be assured by the presence of dG at the 5' end of the full-length cDNA. Furthermore, the cDNA library represents the mRNA population in the cell owing to a bias-free procedure. In this chapter, we describe not only the protocol for preparing the library but also the points for analyzing the 5'-end sequence of the obtained cDNA.

Characterization of the arylsulfatase I (ARSI) gene preferentially expressed in the human retinal pigment epithelium cell line ARPE-19.

PURPOSE: The aim of this study was to characterize the arylsulfatase I (ARSI) gene that has been shown to be preferentially expressed in the human retinal pigment epithelium cell line ARPE-19 and to propose it as a candidate gene responsible for inherited eye diseases such as retinitis pigmentosa (RP). METHODS: Full-length cDNA clones encoding ARSI, arylsulfatase A (ARSA), and sulfatase modifying factor 1 (SUMF1) were isolated from ARPE-19 cDNA libraries constructed using the vector-capping method. The expression vectors for their FLAG-tagged proteins were transfected into ARPE-19 cells, and the expression products were characterized by western blot analysis and arylsulfatase assay. The entire region of the ARSI gene locus was sequenced using the genomic DNA samples of 68 RP patients. RESULTS: Transiently produced ARSI-FLAG was localized to the endoplasmic reticulum and was detected in the cellular fraction and the medium. When ARSI-FLAG and SUMF1-FLAG were coexpressed, the conditioned medium of the transfected cells showed arylsulfatase activity at a range of neutral pH. No mutation was found in the ARSI gene locus of the RP patients examined. CONCLUSIONS: ARSI may be a secreted sulfatase and may function in the extracellular space. Although ARSI may not be a causative gene for lysosomal storage diseases, preferentially expressed in the eye, ARSI would be a candidate gene causing inherited eye diseases for future mutation screening.

Fine expression profiling of full-length transcripts using a size-unbiased cDNA library prepared with the vector-capping method.

Recently, we have developed a vector-capping method for constructing a full-length cDNA library. In the present study, we performed in-depth analysis of the vector-capped cDNA library prepared from a single type of cell. As a result of single-pass sequencing analysis of 24,000 clones randomly isolated from the unamplified library, we identified 19,951 full-length cDNA clones whose intactness was confirmed by the presence of an additional G at their 5' end. The full-length cDNA content was >95%. Mapping these sequences to the human genome, we identified 4,513 transcriptional units that include 36 antisense transcripts against known genes. Comparison of the frequencies of abundant clones showed that the expression profiles of different libraries, including the distribution of transcriptional start sites (TSSs), were reproducible. The analysis of long-sized cDNAs showed that this library contained many cDNAs with a long-sized insert up to 11,199 bp of golgin B, including multiple slicing variants for filamin A and filamin B. These results suggest that the size-unbiased full-length cDNA library constructed using the vector-capping method will be an ideal resource for fine expression profiling of transcriptional variants with alternative TSSs and alternative splicing.

Mutation screening and haplotype analysis of the rhodopsin gene locus in Japanese patients with retinitis pigmentosa.

PURPOSE: To identify nucleotide sequence variations in the rhodopsin (RHO) gene of Japanese patients with retinitis pigmentosa (RP) in order to search for mutations or haplotypes responsible for RP. METHODS: The entire region of RHO locus including a promoter region and introns was sequenced using blood-derived genomic DNA samples donated by 68 patients with RP and 68 control subjects. RESULTS: We found 39 single nucleotide substitutions including 17 rare substitutions of less than 1% in frequency, one insertion/deletion polymorphism, and one CA-repeat polymorphism in a 7.8 kbp region spanning the promoter, five exons, and four introns of the RHO gene locus. There were no affected subjects with amino acid substitutions in RHO, and there was 1 control subject with a novel substitution (Ala42Thr) who had no symptoms of RP. Fine analysis of single nucleotide polymorphism (SNPs) revealed eight haplotype structures of the Japanese RHO locus. There was no significant difference between RP patients and controls in terms of haplotype frequency. CONCLUSIONS: No mutation causing an amino acid substitution of RHO was observed in 68 Japanese patients with RP, but 1 control subject did have a novel amino acid substitution. The Japanese RHO locus is comprised of eight major haplotypes. The RP-associated haplotype was not identified. The haplotype-tagging SNPs identified in this study will be useful as markers for the linkage-based screening of RP patients.

Characteristics of polyclonal anti-human nephrin antibodies induced by genetic immunization using nephrin cDNA.

BACKGROUND: Nephrin is an essential protein for maintaining the normal structure of the podocyte foot process and the glomerular filtration barrier. To analyse the mechanism of proteinuria and treatment of nephrotic syndrome, we produced and characterized polyclonal anti-human nephrin antibodies using a newly developed genetic immunization technique. METHODS: An expression vector with full-length or fragmented cDNA of human nephrin protein was administered to female Lewis rats once a week for 12 weeks using a gene-gun method. Antibody production against different fragments of nephrin protein was then analysed by ELISA, Western blot analysis, immunoprecipitation and FACS analysis. We also analysed recognition of native nephrin protein using immunohistochemistry and electron microscopy, and conducted a functional analysis of in vitro clustering of nephrin protein. RESULTS: Serum anti-nephrin antibody titers reached a maximum level at 8 or 12 weeks. Four of five antibodies induced with cDNA of five different Ig-like motif fragments showed antigen-specific binding to Escherichia coli and produced recombinant human nephrin protein. Only two of these four antibodies, plus one antibody induced by full-length human nephrin protein cDNA, bound to solubilized native nephrin protein. These three IgG antibodies, subclasses IgG1, IgG2a and IgG2b, showed fine granular staining along the glomerular basement membrane of normal human glomeruli and clustering of human nephrin protein on plasma membranes of HEK293 cells. CONCLUSIONS: We successfully produced polyclonal anti-human nephrin antibodies by genetic immunization using nephrin cDNA. These new antigen-specific polyclonal antibodies will be useful for functional analysis and tissue staining of native nephrin protein.

Vector-capping: a simple method for preparing a high-quality full-length cDNA library.

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.

Determination of the capped site sequence of mRNA based on the detection of cap-dependent nucleotide addition using an anchor ligation method.

The sequence analysis of the 5' ends of cDNAs prepared using the anchor ligation method has revealed that most of the full-length cDNAs have an additional dGMP at their 5' end that is absent in the corresponding genome sequence. Using model RNA transcripts with cap analogues possessing 7-methylguanosine and adenosine, the base of the added nucleotide has been shown to be complementary to the base of the cap analogue, suggesting that the cDNAs possessing an additional dGMP are derived from intact mRNAs with the cap structure. On the other hand, cap-free RNA did not produce cDNA with an extra dGMP. These findings suggest that we can determine whether or not the cDNA starts from the capped site sequence of mRNA based on the presence or absence of an additional dGMP at the 5' end of the cDNA synthesized using the anchor ligation method. This approach will be useful to determine the capped site sequence of mRNA, thus, to identify transcription start sites.

DNA immunization via intramuscular and intradermal routes using a gene gun provides different magnitudes and durations on immune response.

We investigated the antibody (Ab) and cytotoxic T lymphocyte (CTL) responses to gene gun (GG)-mediated DNA immunization via the intramuscular (i.m.) and intradermal (i.d.) routes. BALB/c mice were immunized five times at weekly intervals with plasmid DNA encoding enhanced green fluorescent protein (EGFP). EGFP production was rapidly detected in the target tissues after injection via either delivery route. There were significant differences in the magnitude and duration of the Ab and CTL responses according to the route employed. Intradermal injection elicited higher Ab and CTL responses to EGFP than i.m. injection 1 week after the last immunization. However, both immune responses were reduced rapidly 5 weeks after the last immunization via i.d. injection. In contrast, in mice injected via the i.m. routes, Ab and CTL responses 5 weeks after the last immunization remained at levels similar to those detected after 1 week. All mice generated a predominantly IgG1 Ab response via either route. These findings suggest that a combination of these two routes of DNA immunization would provide optimal conditions for induction of a broad immune response, and this information is expected to be very important for future applications of DNA vaccination.

PCIF1, a novel human WW domain-containing protein, interacts with the phosphorylated RNA polymerase II.

Phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAP II) largest subunit has an important role in transcription elongation and in coupling transcription to pre-mRNA processing. To identify proteins that can directly bind to the phosphorylated CTD, we screened a human cDNA expression library using 32P-labeled CTD as a probe. Here we report the cloning and characterization of a novel human WW domain-containing protein, PCIF1 (phosphorylated CTD interacting factor 1). PCIF1 is composed of 704 amino acids. The WW domain of PCIF1 can directly and preferentially bind to the phosphorylated CTD compared to the unphosphorylated CTD. PCIF1 binds to the hyperphosphorylated RNAP II (RNAP IIO) in vitro and in vivo. Double immunofluorescence labeling in HeLa cells demonstrated that PCIF1 and endogenous RNAP IIO are co-localized in the cell nucleus. Thus, PCIF1 may play a role in mRNA synthesis by modulating RNAP IIO activity.

Interaction between mutant ataxin-1 and PQBP-1 affects transcription and cell death.

PQBP-1 was isolated on the basis of its interaction with polyglutamine tracts. In this study, using in vitro and in vivo assays, we show that the association between ataxin-1 and PQBP-1 is positively influenced by expanded polyglutamine sequences. In cell lines, interaction between the two molecules induces apoptotic cell death. As a possible mechanism underlying this phenomenon, we found that mutant ataxin-1 enhances binding of PQBP-1 to the C-terminal domain of RNA polymerase II large subunit (Pol II). This reduces the level of phosphorylated Pol II and transcription. Our results suggest the involvement of PQBP-1 in the pathology of spinocerebellar ataxia type 1 (SCA1) and support the idea that modified transcription underlies polyglutamine-mediated pathology.

The human ribosomal protein genes: sequencing and comparative analysis of 73 genes.

The ribosome, as a catalyst for protein synthesis, is universal and essential for all organisms. Here we describe the structure of the genes encoding human ribosomal proteins (RPs) and compare this class of genes among several eukaryotes. Using genomic and full-length cDNA sequences, we characterized 73 RP genes and found that (1) transcription starts at a C residue within a characteristic oligopyrimidine tract; (2) the promoter region is GC rich, but often has a TATA box or similar sequence element; (3) the genes are small (4.4 kb), but have as many as 5.6 exons on average; (4) the initiator ATG is in the first or second exon and is within plus minus 5 bp of the first intron boundaries in about half of cases; and (5) 5'- and 3'-UTRs are significantly smaller (42 bp and 56 bp, respectively) than the genome average. Comparison of RP genes from humans, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae revealed the coding sequences to be highly conserved (63% homology on average), although gene size and the number of exons vary. The positions of the introns are also conserved among these species as follows: 44% of human introns are present at the same position in either D. melanogaster or C. elegans, suggesting RP genes are highly suitable for studying the evolution of introns.