G) present in 13 of the 235 patients. Only 22% (8/36) of the ASO-amenable variants had been previously classified in ClinVar as pathogenic or likely pathogenic (Extended Data Fig. 3a); 50% (18/36) had been previously classified as likely benign, uncertain significance or conflicting interpretations of pathogenicity; and 28% (10/36) were not reported in ClinVar (as opposed to around 17% for non-ASO-amenable variants). These findings suggest that ASO-amenable variants are often misinterpreted and underrepresented in ClinVar./p>T, c.5763-1050A>G), patient fibroblasts were available for RNA-seq; for nine additional probably/possibly ASO-amenable variants, gDNA samples were available, and experimental minigene splicing assays were successfully established (for more details about minigene assays, see Methods and Supplementary Tables 10–12). All tested variants (11/11) yielded the predicted mis-splicing consequences (for RNA-seq, see the following sections; for minigene assays, see Extended Data Fig. 5). Furthermore, we conducted ASO screens for six probably ASO-amenable variants (two based on patient fibroblasts and four based on minigene assays), and successfully identified ASOs capable of correcting mis-splicing for all six (see the following sections for patient-fibroblast-based screens, Fig. 4 for minigene-based screens and Supplementary Table 9 for a summary)./p>G) and c.2839-581G>A; b, c.6348-986G>T; and c, c.3994-159A>G), small-scale ASO screening was performed, which showed that the mis-splicing events caused by all tested variants can be rescued by ASOs. NT, NT-22 (a non-targeting ASO). Blue asterisks, white (or black) arrows and white (or black) ‘x’ marks indicate bands validated by Sanger sequencing (orange asterisk indicates a band not validated by Sanger sequencing). ΔMES, change in MaxEntScan score when a given variant is introduced. For gel source data, see Supplementary Fig. 1./p>G, c.2250G>A, chr11:108243936-108243949insAlu and c.2639-22_2639-20del were found in thirteen, five, two and two individuals, respectively (Supplementary Table 9). In addition, several different ASO-amenable variants had splicing consequences that appeared addressable with a single ASO; for instance, both c.2839-579_2839-576del and c.2839-581G>A result in the inclusion of the same pseudoexon (Fig. 3). We therefore subdivided variants into ‘treatment groups’, each potentially addressable with a single ASO drug. On the basis of these patterns, around 70% (24/35) of amenable individuals were predicted to be treatable with a total of five different splice-switching ASOs, whereas developing splice-switching treatments for all 35 individuals would require 15 distinct therapeutic ASO drugs (Extended Data Fig. 4b and Supplementary Table 9)./p>G (Fig. 3). The variant, located deep in intron 38, results in the inclusion of a 137-bp pseudoexon, thereby causing a frameshift in the resulting mature product26. This variant is associated with a mild A-T phenotype owing to partial leakiness of its gain-of-splicing effect. It is a founder variant in the UK, with an estimated disease allele frequency of 18% in individuals with A-T in the UK (refs. 27,28). In the ATCP cohort, composed predominantly of individuals from the USA, it was found in a compound heterozygous state in 13 unrelated patients, representing a population frequency of 5.5% (13/235) and a disease allele frequency of 2.8% (13/469)./p>T (Fig. 3). This variant has been previously identified in the homozygous state in patients with classical (severe) A-T. A lymphoblastoid cell line with this variant had no residual protein or enzymatic activity29 (A. M. R. Taylor, personal communication); that is, this variant is a null variant. This variant is predicted to have a benign coding effect (p.Ala2622Val; predicted benign by REVEL), with a pathogenic effect that is mediated by mis-splicing: it creates a strong splice donor site within exon 53 (of 63 exons), causing truncation of the exon by 64 bp, which results in frameshift and subsequent premature translational termination. Inhibition of this splice donor site with a morpholino oligonucleotide has been previously shown to rescue the cellular phenotype of a patient-derived cell line30./p>T was encountered in one individual in the ATCP cohort (DDP_ATCP_520, currently 20 years old). Separately, we also identified a second, younger (one year old at referral; currently six years old) child with A-T with this variant as well (Extended Data Fig. 6a and Supplementary Note 4). Whereas most patients with A-T are diagnosed after the initial onset of symptoms (Supplementary Table 1), this child was diagnosed as an infant on the basis of an abnormally low T cell receptor excision circle (TREC) count. TREC assays are used in newborn screening to identify infants at risk for severe combined immune deficiency (SCID), but have also incidentally identified cases of A-T (ref. 31). Exome sequencing in this child showed compound heterozygosity for two ATM variants: c.7865C>T and c.8585-13_8598del (confirmed by trio Sanger sequencing; Supplementary Table 6). The latter is a 27-bp deletion at the intron–exon junction of exon 59, strongly predicted to cause complete loss of function (Supplementary Note 4). This combination of variants predicted a classical, early-onset A-T phenotype (Fig. 1d)./p>T. PCR with reverse transcription (RT–PCR) and RNA-seq analysis of splicing patterns in this cell line showed an abnormal truncation of exon 53 owing to premature splice donor site usage, consistent with previous studies30 (Extended Data Fig. 6b,c). Analysis using allele-specific PCR primers (designed to exclude the non-target c.8585-13_8598del allele) showed that ATM mis-splicing by c.7865C>T is complete, without detectable leakiness (Extended Data Fig. 6d)./p>T. Biologically independent experiments (independent transfections) were conducted (n = 3, initial; n = 2, fine-tuning). For some ASOs in the fine-tuning screening, the first two letters are omitted. Error bars, mean ± 95% confidence interval (shown only for conditions with n ≥ 3). A one-sample two-tailed t-test was used to assess statistical significance; means were compared to a constant value of 0 because no background normal splicing was observed in cells that were mock-transfected or transfected with non-targeting ASOs. *P < 0.05; **P < 0.01. AT010, *P = 0.0441; AT004, *P = 0.0240; AT001, *P = 0.0453; AT002, **P = 0.0010; AT005, **P = 0.0093; AT006, **P = 0.0060; AT007, **P = 0.0001; AT008, **P = 0.0082. Four top-performing ASOs (blue letters) were selected for further validation (b,c). For RT–PCR gel source data, see Supplementary Fig. 1. b, ASO-mediated restoration of irradiation-induced ATM signalling in patient fibroblasts, measured by immunoblotting. 07, 08, 22 and 26 represent AT007, AT008, AT022 and AT026, respectively; NT, NT-22 (a non-targeting ASO). pP53, phospho-P53; pKAP1, phospho-KAP1. Biologically independent experiments (independent transfections) were conducted: pP53 (n = 2, hypomorphic cases ± irradiation; n = 4, AT022, AT026; n = 5, the other conditions), pKAP1 (n = 4, AT007 and AT022; n = 5, the other conditions). Error bars, mean ± 95% confidence interval (shown only for conditions with n ≥ 3). A two-sample (comparing each condition to NT-22) two-tailed t-test was used for statistical analysis. *P < 0.05; **P < 0.01. For pP53, AT007, **P = 0.0024; AT008, **P = 0.0001; AT022, **P = 0.0471. For pKAP1, AT008, *P = 0.0201; AT022, *P = 0.0175; AT026, **P = 0.0073. Representative blot images are shown in Extended Data Fig. 8a. For blot source data, see Supplementary Fig. 1. c, ASO-mediated restoration of irradiation-induced ATM signalling in patient fibroblasts, measured by immunofluorescence staining. Scale bar, 50 μm. For a quantitative summary of the complete results, see Extended Data Fig. 8b./p>G variant, supporting the potential clinical relevance of this rescue./p>G variant. A fibroblast cell line was established from a patient with A-T from the ATCP cohort (DDP_ATCP_42) and used to confirm the mis-splicing consequences of c.5763-1050A>G (Fig. 3 and Extended Data Fig. 9). ASOs were designed to block the pseudoexon usage associated with this allele, and screening in patient fibroblasts successfully identified a lead ASO that was capable of rescuing ATM function (Extended Data Figs. 10 and 11, Supplementary Note 5 and Supplementary Figs. 9 and 11)./p>T-targeting ASO AT008 (renamed atipeksen) was selected for further clinical development. It was chosen because of the association of c.7865C>T with severe disease (classical A-T), the robustness of atipeksen-mediated RNA and cellular functional rescue and the opportunity for early therapeutic intervention before the onset of major neurological morbidity in the previously identified young child with this variant. (Note that the other identified individual with this variant, DDP_ATCP_520, was not considered to be a suitable candidate for clinical intervention owing to the advanced stage of the disease)./p>T who has been treated with AT008 (atipeksen), and individuals in the ATCP cohort, who were enrolled for WGS variant call validation by Sanger sequencing and mis-splicing validation by minigene assay and RNA-seq. gDNA samples extracted from the saliva of patients were provided by the Broad Institute. Whole-blood samples were provided by their physicians through the ATCP foundation, and RNA samples were extracted from these./p>T (p.Glu1978Ter); this variant has the highest allele frequency in this ATCP cohort among the variants annotated as pathogenic in ClinVar. It has gnomAD v.3.1 and ATCP cohort allele frequencies of 0.0000349045 and 0.034 (16/470), respectively. For the variant calls that had passed the allele frequency filter, their protein-coding and splicing impacts were examined on the basis of multiple computational tools: REVEL (for protein-coding impacts) and SpliceAI and MaxEntScan (for splicing impacts). Missense variants that were predicted as pathogenic by REVEL (score ≥ 0.5) were considered as disease candidate variants. Mis-splicing events with a SpliceAI score of 0.1 or higher were considered as likely true events. If the consequence of the mis-splicing is predicted to result in frameshift or loss of a crucial domain of the protein, the variant that caused the mis-splicing was classified as a disease candidate variant. For the patients in whom fewer than two disease candidate events were identified up to this step, we reviewed the remaining variants on a case-by-case basis (Supplementary Note 2)./p>T) who has been under treatment with atipeksen, as well as on five individuals in the ATCP cohort (four families; DDP_ATCP_42 (with c.5763-1050A>G), DDP_ATCP_218, DDP_ATCP_38/39, DDP_ATCP_96). In all six cases, we confirmed with Sanger sequencing that the two disease candidate variants in each case are in trans (Supplementary Tables 1 and 6)./p>3 nt by the variant]./p>
0.5)./p>
T [in DDP_ATCP_138] and c.4801A>G [in DDP_ATCP_302]) did not pass this criterion as they showed predominant skipping of the exon of interest even in the absence of the variant of interest in the ATM gene region of the plasmids./p>T, a total of 32 ASOs were designed (12 for the initial screening and 20 for the fine-tuning screening). The ASOs were designed to be complementary to either the region encompassing the novel splice donor site in exon 53 created by c.7865C>T or predicted splice silencers surrounding the exon 53 canonical splice donor site. These silencers were predicted on the basis of a previously published hexamer-based model62. For c.5763-1050A>G, a total of 27 ASOs were designed (12 for the initial screening and 15 for the fine-tuning screening) to be complementary to the regions encompassing the novel splice donor site in intron 38 created by c.5763-1050A>G, the cryptic acceptor site of the pseudoexon in intron 38 or predicted splice silencers within the pseudoexon (also predicted on the basis of the hexamer model). For minigene-based validation of ASO amenability, a total of 24 ASOs were designed for 4 ASO-amenable variants (c.2839-579_2839-576del, c.2839-581G>A, c.6348-986G>T and c.3994-159A>G). The ASOs were designed to block either the splice donor/acceptor site or predicted exonic splicing silencers within a pseudoexon of interest. NT-20 and NT-22 (non-targeting oligonucleotides with the same chemistry) were used as negative controls1. For in vitro toxicity testing, ASO-tox, a gapmer with known toxicity, was used. All ASO sequences and detailed chemical modifications of ASOs are provided in Supplementary Table 13. All ASOs were manufactured by Microsynth. The ASO drug substance used in the atipeksen N-of-1 clinical trial was manufactured by ChemGenes in accordance with GMP guidelines./p>G, the distance between the two ATM variants was too far (around 2 kb) to distinguish the two bands representing normally and abnormally spliced products (which differ by 137 bp) on a agarose gel; therefore, a nested PCR was performed. PCR was performed using 1 µl of cDNA and a standard condition (35 cycles; 98 °C for 5 s, 60 °C for 15 s, 72 °C for 45 s). Relative quantities of the normally and abnormally spliced transcripts were measured by 1.5% agarose gel electrophoresis and densitometry analysis using ImageJ./p>