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Download Phenotypic variability of osteogenesis imperfecta is not accounted
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Phenotypic variability of osteogenesis imperfecta is not accounted for by disruption of splicing cis-elements +1Kaneko, H; 1Kitoh, H, 1Matsuura, T; 1Masuda, A; 1Ito, M; 2Mottes, M; 3Rauch, F; 1Ishiguro, N; 1Ohno, K +1Nagoya University Graduate School of Medicine, Nagoya, Japan, 2University of Verona, Verona, Italy, 3 Shriners Hospital for Children and McGill University, Montréal, Québec, Canada [email protected] INTRODUCTION: Osteogenesis imperfecta (OI) is caused by mutations in COL1A1 on chromosome 17 or COL1A2 on chromosome 7. Similar mutations in each gene exhibit widely variable phenotypes, and genotype-phenotype correlations have not been fully elucidated. Pre-mRNA is regulated by both intronic and exonic splicing ciselements. Both constitutively spliced and alternatively spliced exons harbor exonic splicing enhancers (ESEs) and silencers (ESSs). A mutation in the coding region is generally predicted to exert its pathogenicity by compromising a functional amino acid, but nonsense, missense, and even silent mutations potentially disrupt an ESE or ESS, thereby resulting in aberrant splicing. More than 16% to 20% of exonic mutations are predicted to disrupt an ESE. Here, we hypothesized that preservation or disruption of an exonic splicing cis-element determines the clinical phenotype. In a Japanese family, hyperuricemia cosegregated with OI. In order to trace a responsible gene for hyperuricemia, we looked for a founder haplotype shared by the Japanese family and the previously reported Italian and Canadian families with COL1A1 c.3235G>A. METHODS: Informed consent was obtained from all family members. The current studies have been approved by the Institutional Review Boards of the Nagoya University, the University of Verona, and the McGill University. We performed mutation analysis of a Japanese family with OI type I using genomic DNA and cDNA. Five missense mutations (c.3226G>A, c.3226G>T, c.3235G>A, c.3244G>T, and c.3253G>A) in exon 45 of COL1A1 cause mild to lethal OI phenotypes. We predicted effects on pre-mRNA splicing of 18 sequence variants with or without each mutation in the presence or absence of each of two SNPs (rs1800215 and rs1800217) in exon 45 of COL1A1 using five web-based programs: ESEfinder 3.0, ESRsearch, FAS-ESS, PESXs, and RESCUE-ESE. We constructed 18 variant COL1A1 minigenes including exons 44-46 and the intervening introns using the pcDNA3.1(+) mammalian expression vector (Fig. 2A). We transfected each of minigene constructs into HEK293 cells. After 48 hours, we extracted total RNA and synthesized cDNA. We performed RT-PCR using generic primers on pcDNA3.1. To determine haplotypes of the Japanese, Italian, and Canadian families with COL1A1 c.3235G>A, we genotyped three microsatellite markers (D17S1293, D17S1319, and D17S788) and a SNP (rs2075554) in intron 11 of COL1A1. RESULTS: A father and his three sons had blue sclera, dentinogenesis imperfecta, and joint laxity (Fig. 1). Two sons (II-1 and II-3) had histories of more than 10 fractures before age 13 years, but three other members experienced no fracture. One son (II-1) had hearing loss from age 10 years and hip joint deformities due to repeated femoral fractures. We identified a dominant missense mutation, c.3235G>A in COL1A1 exon 45, in four patients in the Japanese family (Fig. 1). We asked if some mutations in COL1A1 exon 45 disrupt an exonic splicing cis-element and result in phenotypic variability. Three programs showed that five mutations and two SNP in exon 45 were predicted to affect 16 putative splicing cis-elements. We analyzed 18 minigenes, and found that none affected pre-mRNA splicing (Fig. 2B). In the Japanese family, hyperuricemia cosegregated with OI, but not in the previously reported Italian and Canadian families with c.3235G>A. We found that each family carried a unique haplotype (Fig. 1). Thus, hyperuricemia was possibly caused by a mutation in a gene on the same chromosome as COL1A1. Two candidate genes are on the same chr as COL1A1 at 17q21.31-q22. PRPSAP1 on chr 17q24-q25 encodes PAP39 and PRPSAP2 on chr 17p12-p11.2 encodes PAP41. PAP39 and PAP41 are subunits of phosphoribosylpyrophosphate synthetase that leads to urate production. Sequencing the entire coding regions of both genes, however, revealed no mutation. DISCUSSION: Patients within the same family carrying the same mutation sometimes exhibit variable OI phenotypes. In the Italian family with the c.3235G>A mutation, the son suffered from many fractures, but his mother did not have any. In the Canadian family with the same mutation, the father was classified as OI type IV, whereas his daughter was OI type I. Variable clinical phenotypes of the c.3235G>A mutation is potentially due to differences in environmental factors or to SNPs in disease-modifying genes. Phenotypic variability of mutations in COL1A1 exon 45 is not accounted for by splicing dysregulations. Our analysis suggests that the currently available algorithms cannot efficiently predict ESEs. To our knowledge, association of hyperuricemia with OI has been reported in a single family, in which two of three OI patients had gouty arthritis and hyperuricemia at young ages by Allen et al. (1955). The cosegregated hyperuricemia is likely to have arisen from a mutation in a yet unidentified gene on chromosome 17 in the Japanese family. Figure 1. OI pedigrees and haplotypes of the Japanese, Italian and Canadian families with the c.3235G>A mutation of COL1A1. The clinical subtypes are all type I except for the Canadian father (V-1) who exhibits type IV. The Japanese, Italian, and Canadian families carry their unique haplotypes (shown by solid, dotted, and broken boxes, respectively) that are not shared across different ethnic origins. The D17S1293 genotype in the Canadian family is not informative. Figure 2. Splicing assay using COL1A1 minigenes. (A) Schematic representation of COL1A1 minigenes. (B) RT-PCR of minigenes introduced into HEK293 cells. All constructs show a single fragment of 336 bp, indicating that COL1A1 exon 45 is not skipped in any constructs. SNP1 and SNP2 are rs1800215 and rs1800217, respectively. Untransfected cells are used as a negative control (NC). Poster No. 559 • 56th Annual Meeting of the Orthopaedic Research Society