Relationship between visual field loss and maximal combined electroretinographic responses in retinitis pigmentosa : comparison among genetically different types

1984年から1996年までに千葉大学眼科を受診した定型網膜色素変性症228例について常優26例, 常劣64例, 孤発例138例に分け動的量的視野および網膜電図を検討した。Goldmann視野におけるV-4イソプターでは5から10cm^2までと150から250cm^2までの2群に別れ, 1-4イソプターでは5cm^2以下の群のみ認められた。加齢及び遺伝形式による差異は認められなかった。網膜電図ではa, b波ともに正常対照群より減少してはいるものの, 遺伝形式による差異は認められなかった。また, 網膜電図で振幅を認められる割合はV-4イソプターおよびI-4イソプターの面積と相関していることが示唆されたが, 遺伝形式による差異は認められなかった。網膜電図の振幅の比であるb/a比は正常対象群に対して疾患群は減少していたが, 常備は特に他に比べ有意に減少していた。定型網膜色素変性症の網膜電図や視野の検討は数多くなされてきたが, b/a比について統計的考察がなされてきたことはない。a波およびb波は組織学的に発生起源が異なっており, b波はa波のインパルスによって二次的に引き起こされることは以前より知られてきている。網膜電図において常備のb/a比が有意な低下を示すことは, 網膜障害の機序が他と異なる可能性が示唆された。Analyses were performed on 228 Japanese patients with retinitis pigmentosa (RP) who were classified with autosomal dominant (ADRP, n=26), autosomal recessive (ARRP, n=64), and simplex (simplex RP, rc=138) inheritance. Visual fields were tested with Goldmann perimetry. Maximal combined responses of electroretinogram (ERG) with 20-Joule white flash stimulation were recorded after dark adaptation for 20 minutes. The visual field with the V-4 isopter demonstrated two unique groups, represented by dense areas between 5 and 10cm^2 and between 150 and 250cm^2, while only one unique group was observed within the 5cm^2 area with the 1-4 isopter. No age or inheritance type of effect was seen. A-and b-wave amplitudes were equally low in the 3 groups, as compared with normal subjects. The b/a ratio was significantly smaller in the ADRP group, compared with the others. The rate of detectable ERG responses decreased as the visual field became smaller. There was no inheritance effect. A lower b/a ratio in ADRP patients suggested that retinal functional abnormalities differed from ARRP and simplex RP patients

Additional file 1: Table S1. of Non-coding single nucleotide variants affecting estrogen receptor binding and activity

The list of all ER ChIP-seq datasets in breast cancer. Table S2. List of E2-regulated genes common in vitro, in vivo, and TCGA. Table S3. List of the studies used for the identification of E2-regulated genes. Table S4. Primer sets used for different assays. Table S5. The list of regulatory SNVs in MCF7 Cell line. Table S6. The list of regulatory SNVs in BT474 cell line. Table S7. The list of regulatory SNVs in MDA-MB-134 cell line. Table S8. The list of regulatory SNVs in T47D cell line. Table S9. The list of regulatory SNVs in TAMR cell line. Table S10. The list of regulatory SNVs in ZR75 cell line. Table S11. The list of regulatory SNVs in good prognosis tumors. Table S12. The list of regulatory SNVs in bad prognosis tumors. Table S13. The list of regulatory SNVs in metastatic tumors. Table S14. The allele frequency of top RegSNVs in ER-binding sites with sufficient coverage. (XLSX 3641 kb

Additional file 2: Figure S1. of Non-coding single nucleotide variants affecting estrogen receptor binding and activity

The UCSC gnome browser view of the second intron in IGF1R gene. The index SNP, rs62022087, seems to be located in a region bound by several chromatin-modifying factors based on ENCODE data. Figure S2. The visualization of ChIP-seq reads from multiple cell lines over rs62022087 SNP site in two individual studies: (A) Hurtado et al. {Hurtado, 2011 #23}, (B) Joseph et al. {Joseph, 2010 #15}. Figure S3. The distribution of RegSNVs over the gnome across a panel of breast cancer cell lines, good and bad prognosis tumors. The binding sites from different ER ChIP-seq datasets were extracted and annotated based on their location in the genome. The majority of the binding sites are located in the intergenic and intronic areas. (DOCX 384 kb

Additional file 2: Figure S1. of Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models

Sanger sequencing shows the insertion of Y537S (A > C) and D538G (A > G) in T47D and MCF7 cells. Figure S2 Total ER and phospho ER blotting in all clones of T47D and MCF7 cell lines. a Quantification of P-ER(S118) bands from three independent experiments. Band densities were calculated by ImageJ. P-ER values were corrected to total ER level, and then normalized to vehicle-treated WT groups. b T47D and MCF7 WT or mutant individual clones were hormone-deprived and treated -/+ 1 nM of E2 for 24 hand IB was performed for ER and p-ER at Ser118 site. B-actin was used as a loading control. c Post-hormone-deprived MCF7 or T47D clones were treated with 1 nM of E2 combined with or without 1 μM of Ful for 24 h. RT-qPCR was done using PGR primers. One-way Anova was performed between the basal expression of PGR in each mutant clone and the average expression of PGR in the WT clones (*p < 0.05, **p < 0.01, red) and Student’s t test was used to compare the response before and after fulvestrant treatment (*p < 0.05, **p < 0.01, black). Figure S3 Lack of significant AR overexpression in MCF7 and T47D ESR1-mutant cells: log2 TPM expression of AR in MCF7 and T47D cells based on RNA-seq experiment. b The post-hormone-deprived MCF7 or T47D cells (pooled) were treated with 1 nM E2 combined with or without 1 μM of fulvestrant (ICI) for 24 h. RT-qPCR was done using AR-specific primers. b Immunoblots of AR expression (CST #5153) in post-hormone-deprived MCF7 or T47D cells. Experiments were performed three times, and AR expression was quantified; bars present average AR expression in mutant relative to WT cells. One-way Anova was performed comparing AR mean expression in each mutant clone with mean expression in the WT clones (ns). Figure S4 The ligand-independent growth of T47D-Y537S clones depends on charcoal-stripped serum (Gibco #12676 serum was used in this experiment). WT or mutant clones were hormone-deprived for 3 days, pooled, and treated with veh or 1 nM E2 for up to 9 days. Figure S5 Dose–response curves for 2D growth were plotted for Y537S and D538G mutants of T47D (a) and MCF7 (b) cells after hormone deprivation for 3 days. The cells were treated with 20 pM E2 + Ful, AZD9496, 4OHT and raloxifene. The dose–response curves were fitted with a nonlinear regression model in GraphPad Prism. This figure is a representative of one individual experiment that was repeated six times with consistent results. All experiments were performed in six biological replicates. Figure S6 PCA analysis of 1000 top variable genes between WT and mutants. The top 1000 most variable genes were selected based on interquartile range. The PCA analysis was performed and plotted using PCA function in R. Figure S7 Heatmap of variable genes (Anova, p < 0.0005, maximum FC >2) in mutants and WT cells. Gene expression TPM was estimated by Salmon package. Anova was then used to identify genes differentially expressed between the samples. Genes with a p value <0.0005 and FC >2 that were differentially regulated in at least one mutant vs WT-veh were selected for this heatmap. Figure S8 The post-hormone-deprived MCF7 or T47D cells (pooled or individual clones) were treated with 1 nM of E2 -/+ 1 μM of Ful for 24 h. RT-qPCR was done using GREB1 (a) or IGFBP4 (a) primers. All experiments were performed in three biological replicates. One-way Anova was performed between the basal expressional levels in each mutant clone and the average expression of GREB1 and IGFBP4 in the WT clones (*p < 0.05, **p < 0.01, red) and Student’s t test was used to compare the response before and after Ful treatment (*p < 0.05, **p < 0.01, black). Figure S9 Log2 TPM expression of PGR, GREB1 and IGFBP4 levels in MCF7 and T47D cells based on the RNA-seq experiment. Figure S10. The post-hormone-deprived MCF7 or T47D cells (pooled or individual clones) were transfected with scramble siRNA or ESR1 siRNA for 24 h, and then treated -/+ 1 nM of E2 for 24 h. RT-qPCR was done using ESR1, PGR, or IGFBP4 primers. All experiments were performed in three biological replicates (one-way Anova, *p < 0.05; **p < 0.01). Figure S11 Overlap of novel ligand-independent regulated genes of the ESR1 mutations within one cell line (a) and between the cell lines (b) (chi-square test, **p < 0.01). (PDF 2550 kb

Additional file 1: Table S1. of Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models

The sequence of sgRNA and oligos used to generate T47D ESR1 mutant cell lines via CRISPR. Table S2 DNA sequence of the oligos used to generate MCF7 ESR1 mutant cell lines via AAV. Table S3 Sequence of the primers used for qPCR assay. Table S4 List of all ligand-independent genes differentially regulated in mutant cells vs WT (FC >2, p < 0.005). Table S5 Disease and function pathways enriched in mutant cells in the absence of estrogen. The novel ligand-independent genes, which were differentially regulated in mutants of each cell line, were pooled and submitted for IPA pathway analysis. The top five relevant functions that were statistically significant are presented in this table. (ZIP 266 kb