ATCP的全球A-T家族数据平台提供了235例A-T患者的WGS和临床数据 。我们对数据的访问已由ATCP数据访问委员会批准。 　　在波士顿儿童医院（10-02-0053）的机构审查委员会（IRB）的批准下，选定的A-T患者在曼顿孤儿疾病研究中心招募
。其中包括已接受AT008（Atipeksen）治疗的C.7865c> t的个人 ，以及ATCP队列中的个体，他们通过Minigene Assay和RNA-Sequs招募了通过Sanger测序进行WGS变体呼叫验证的人。从患者的唾液中提取的GDNA样品由Broad Institute提供 。其医生通过ATCP基础提供了全血样品
，并从中提取了RNA样品
。 　　使用来自患者及其家族的细胞的功能研究是在波士顿儿童医院的曼顿孤儿疾病研究基因发现核心维护的IRB批准方案的主持下进行适当同意后进行的。皮肤成纤维细胞源自使用外植物培养物从患者的皮肤中进行的2毫米打孔活检 。维持成纤维细胞并将其传递在含有10％胎牛血清（Gibco）的DMEM（Fisher Scientific）的培养基中
。实验中使用的成纤维细胞在通过20下。 　　使用BWA（v.0.7.17）（参考文献46）将WGS读取与GRCH38/HG38对齐，并根据GATK最佳实践Workflows12进行预处理和质量控制 。Multiple computational tools were used to call diverse types of variants, including GATK-HaplotypeCaller (v.3.5) (ref. 12), VarScan2 (v.2.4.4) (ref. 13) and Strelka2 (v.2.9.10) (ref. 14) for SNVs and short indels (less than 50 nt in length; Supplementary Table 2) and Delly (v.0.8.6) (ref. 15), Pindel（v.0.2.5b8）（参考文献16） ，熔体（v.2.2.2）（参考文献17）和XTEA（v.0.1.7）18用于SVS（长度为50或更多NT；补充表3）
，熔体和XTEA用于转座子
。对于具有不精确边界的大型CNV，我们手动检查了综合基因组观看器（IGV; v.2.8.9）（参考文献47）的读取对齐 ，以确定变体的粗糙边界。 　　为了分析ATCP队列中235个人之间的相关性
，我们将VCFTools（V.0.1.17）与“相关性2 ” Option48（补充表1）（基于KING软件包49）使用 。对于具有注释相关性信息的个体，在临床记录中 ，所有注释都与推断的相关性一致
。 　　对于变体效应预测
，使用VEP（版本100）50来注释所有SNV和缩写indels。使用REVEL21和文献中蛋白质功能的实验证据评估了蛋白质编码的影响（补充表7）；使用MaxentsCan24，SpliceAi20和LabRanchor25评估了错误的影响 。下载了GRCH37/HG19上的LabRanchor预测的分支坐标。将它们转换为GRCH38/HG38坐标（使用提升）后 ，检查了它们的潜在重叠和与变体的距离
。对于短于50个核苷酸的变体
，在GNOMAD（v.3.1）（参考文献51）中查找等位基因频率并上面（Freeze 8;参考52）。对于SVS，在DBVAR53 ，DGV54和GNOMAD SVS（v.2.1）（参考文献55）中查找了等位基因频率 。在Clinvar中查找了变体的临床意义（截至2020年6月2日；参考文献56）
。通过手动检查IGV上的原始测序数据，确认了所有疾病候选SV事件。 　　根据ACMG19,57概述的指南
，使用五层系统对疾病候选变体进行了分类 。对于预测的功能丧失变体，我们使用专门的ACMG建议应用PVS1标准58
。对于CNV ，我们使用了ACMG和临床基因组资源（Clingen）提出的专门评分框架59。 　　首先
，我们定义了SDV 。其中包括移封，定格 ，起始损坏和脱夹站点的变体
，以及影响一个或多个外显子的结构变体，以及带有先前功能性损失的实验证据的错义变体和短框架内indels
。将脱夹站点拆除的变体定义为：（在规范剪接位点 ，spliceai供体/受体损失评分≥0.1）和（Maxentscan供体/受体评分
，具有ALT等位基因< MaxEntScan donor/acceptor score with the REF allele) AND [(MaxEntScan donor/acceptor score with the ALT allele < 2) OR (MaxEntScan donor/acceptor score with the ALT allele < 0.3 × MaxEntScan donor/acceptor score with the REF allele)]. All SDVs were considered as disease candidate variants. Second, variants that were annotated as pathogenic or likely pathogenic in ClinVar were considered as disease candidate variants. 　　For the patients in whom fewer than two disease candidate events were identified in the previous two steps, we analysed the remaining variants in each patient on the basis of the population and cohort allele frequencies. We filtered out variant calls whose population or cohort allele frequencies are higher than that of c.5932G>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). 　　Sanger sequencing validation of a subset of disease candidate variants was performed using available patient gDNA samples. The PCR protocol comprised 10 ng template DNA, 10 µl KAPA2G Robust HotStart ReadyMix (2X; Kapa Biosystems), 1 µl site-specific primer pairs (10 µM), and PCR-grade water to a final volume of 20 µl. The cycling parameters were 94 °C for 3 min; 30 cycles of 94 °C for 15 s, 60 °C for 15 s, 72 °C for 15 s; 72 °C for 3 min; and held at 4 °C. Validation primers are listed in Supplementary Table 4. All PCR amplicons were visualized on 2% agarose gels. Variants and corresponding genotypes were confirmed by Sanger sequencing (Supplementary Table 5). 　　Trio Sanger sequencing was performed on the family of the patient (with c.7865C>T）在Atipeksen以及ATCP队列中的五个人（四个家族； DDP_ATCP_42（带有C.5763-1050A> G），DDP_ATCP_218 ，DDP_ATCP_38/39
，DDP_ATCP_ATCP_ATCP_96）中。在所有六种情况下，我们都通过Sanger测序确认 ，每种情况下的两个疾病候选变体都在反式（补充表1和6）中 。 　　在32例（32个家庭）中，发现候​​选疾病的变异是纯合的
。在其他五个病例（五个家庭）中
，疾病候选变体似乎是纯合的，因为在一个基因座的删除与变体重叠（补充表1）。 　　当两个候选疾病变体之间的距离短于读取长度时 ，可以使用基于读取的相位方法来逐步逐步分阶段 。我们使用Whathap（V.1.0）（参考文献23）
，一种基于读取的相位工具，分析了此类案例 ，发现在两种情况（一个家庭）中
，两种疾病候选变体是在反式的。这两个变体仅分开62 bp，也通过手动检查IGV的原始测序数据来证实（补充表1）
。 　　GNOMAD变体共存在数据库可用于预测两个变体可能在顺式或Trans60中。如果两个变体处于相同的单倍型（即顺式）中 ，则它们倾向于出现在同一个体中 。该分析只能针对在GNOMAD数据库中表示的两个候选变体的个体（v.2.1.1
，在GRCH37/HG19坐标中），其全球等位基因频率高于0％ ，小于5％
。ATCP队列中共有47个人（38个家庭）符合这些标准。分析表明
，在所有47个个体中，2种疾病候选变异很可能具有不同的单倍型（补充表1） 。 　　（1）如果变体损坏了规范的剪接位点和蛋白质编码函数 ，则更严重的损害被认为是变体的代表性损害
。（2）关于变体（如果有）的误解或编码影响的可靠实验证据，可以覆盖计算预测。有关分类法的示意图
，请参见图2 。 　　（1）严重：（i）在规范剪接位点处的spliceai供体/受体损失评分≥0.1，（ii）Maxentscan供体/受体评分 ，在该地点的Alt等位基因 < MaxEntScan donor/acceptor score with the REF allele at the site, AND (iii) [MaxEntScan donor/acceptor score with the ALT allele at the site < 2] OR [MaxEntScan donor/acceptor score with the ALT allele at the site < 0.3 × MaxEntScan donor/acceptor score with the REF allele at the site]. 　　(2) Moderate: (i) NOT severe (as defined above), (ii) SpliceAI donor/acceptor loss score at a canonical splice site ≥ 0.1, AND (iii) [MaxEntScan donor/acceptor score with the ALT allele at the site < MaxEntScan donor/acceptor score with the REF allele at the site, MaxEntScan donor/acceptor score with the ALT allele at the site ≥ 2, AND MaxEntScan donor/acceptor score with the ALT allele at the site ≥ 0.3 × MaxEntScan donor/acceptor score with the REF allele at the site] OR [The variant is ≤3 nt away from the LaBranchoR-predicted branchpoint OR the distance between the LaBranchoR-predicted branchpoint and the site is changed by >3 nt by the variant]. 　　(3) No to little: NEITHER severe NOR moderate (as defined above). 　　(1) Severe: (i) frameshift, stop-gain, or start-loss variant OR (ii) missense variant predicted as pathogenic by REVEL (score >0.5）
。 　　（2）否至少：（i）不严重和（ii）由Revel预测为良性的同义变体或错义变体（得分≤0.5）。 　　（1）误解（增益）的增益：（i）spliceai供体/受体增益分数在非典型地点≥0.1和（ii）Maxentscan供体/受体评分且位置等位基因≥2 。 　　（2）EXON跳过或内含子保留（跳过或保留）：任何规范地点的spliceai供体/受体损失评分≥0.1≥0.1 < 0.1 at any non-canonical splice site). 　　(3) Neither: NEITHER gain, skipping, NOR retention. 　　To generate a minigene, we used the pSpliceExpress plasmid, which was a gift from S. Stamm (Addgene plasmid 32485; http://n2t.net/addgene:32485; RRID: Addgene_32485; ref. 61). The genomic fragment with a variant of interest was cloned into the pSpliceExpress donor vector using the BP recombination reaction. The inserted fragments for reference and alternative alleles were generated by a two-step PCR procedure. In the first round of PCR, the genomic region of interest was amplified from patient gDNA with attB tagged primers, which added 12 nucleotides of the attB1 and attB2 sites to the ends of amplicons. The second PCR reaction used the first PCR products as templates and extended them to contain complete attB sequences using universal adapter primer pairs. All PCR reactions were performed with Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific) or PrimeSTAR GXL DNA polymerase (Takara Bio). Primer sequences used for minigene constructions were listed in Supplementary Table 10. Full attB PCR products were purified using the PureLink PCR Purification Kit or PureLink Quick Gel Extraction Kit (Invitrogen). Gateway BP Clonase II Enzyme Mix (Invitrogen) was used to recombine attB PCR products into pSpliceExpress. In brief, approximately 25 fmol (1 kb PCR product is 0.65 ng fmol−1) of purified attB PCR product was added to 75 ng of donor vector, TE buffer and 1 µl of BP Clonase Enzyme Mix to a final reaction volume of 5 µl. The reaction was incubated at room temperature for 1 h, after which 0.5 µl Proteinase K was added to stop the reaction. One microlitre of each BP Clonase reaction product was transformed into 25 µl OneShot TOP10 Chemically Competent Escherichia coli (Thermo Fisher Scientific). Transformed E. coli was spread on LB agar plates with ampicillin (1× LB agar with 50 µg ml−1 ampicillin) and incubated overnight at 37 °C. To screen for positive colonies containing the desired plasmids, a dozen colonies for each variant were picked up and diluted in 50 µl sterile water. Subsequently, colony PCRs were performed using Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific), followed with 2% agarose gel inspection. The cycling programme was: bacteria were lysed and DNA was denatured at 98 °C for 10 min, followed by 30 cycles of 98 °C for 10 s, optimal annealing temperature for 20 s and 72 °C for 30 s, and final extension for 5 min at 72 °C. Primer sequences used for colony PCR are listed in Supplementary Table 10. Positive colonies were inoculated in liquid LB with ampicillin (1× LB and 50 µg ml−1 ampicillin) and were cultured in a shaking incubator at 275 rpm at 37 °C for 12–18 h. Plasmid DNA was extracted from overnight cultures using PureLink Quick Plasmid Miniprep Kit (Invitrogen) or ZR plasmid Miniprep Kit (Zymo Research). The genotypes and the sequences of plasmid inserts were confirmed by Sanger sequencing (Supplementary Table 11). At least one wild-type and one mutant plasmid were identified for each variant. 　　In some variants, full attB PCR products could not be amplified directly from patient gDNA owing to low quality or unavailability of the patient gDNA. In these cases, a wild-type fragment was amplified from human male gDNA (Promega) and used to construct reference plasmids as described above. The Q5 site-directed mutagenesis kit was used to introduce the variants into the reference plasmids (New England Biolabs). Twenty-five-microlitre PCR reactions were set up with mutagenic primers (Supplementary Table 10) and Q5 Hot Start High Fidelity 2X Master Mix to introduce the variant into the reference plasmids and amplify the mutant plasmids. The samples were denatured at 98 °C for 30 s and subjected to 25 cycles of 98 °C for 10 s, 50–72 °C (various annealing temperatures were tested) for 10 to 30 s, 72 °C for 20–30 s per kb, followed by a final extension at 72 °C for 2 min. The linear PCR products were ligated into the plasmid through DpnI restriction digestion and ligation. The mutant plasmids were transformed into competent E. coli. Single colonies were screened and inoculated in liquid LB and ampicillin. Plasmid DNA was collected from overnight cultures. 　　Around 1 × 105 HEK293T cells were seeded in 24-well plates. When the cells reached about 90% confluency, they were transfected using Lipofectamine 3000 (Thermo Fisher Scientific). For each transfection, 4 µl of plasmid was added to each well along with 1.5 µl Lipofectamine, 2 µl P3000 and 50 µl Opti-MEM (Thermo Fisher Scientific). For some transfections, ASOs were also added at a final concentration of 200 µM. Twenty-four hours after transfection, total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). RNA was then reverse-transcribed into cDNA in a 4-μl total reaction consisting of 3 µl RNA and 1 µl of SuperScript IV VILO Master Mix (Thermo Fisher Scientific). The reverse transcription reactions were incubated at 25 °C for 10 min, 50 °C for 10 min and 85 °C for 5 min. To detect transcripts transcribed from the transfected plasmids, 1 µl cDNA was amplified using Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific), 2× KAPA SYBR Fast qPCR Master Mix (Kapa Biosystems) or 2× KAPA HiFi HotStart ReadyMix (Kapa Biosystems). For primers, we used rat insulin primers that bind to the minigene exons flanking the inserted ATM gene region (Supplementary Table 10). The final PCR products were run and visualized on 2% agarose gel. Mis-splicing bands were extracted using the PureLink Quick Gel Extraction (Invitrogen) and confirmed by Sanger sequencing (Supplementary Table 12). 　　If the amount of the canonical splicing isoform represented less than 50% of the total amount of all ATM isoforms, we disqualified and excluded the minigene assay plasmids for further analysis. We found that some of the plasmids bearing the ATM gene region did not express the normally spliced isoform even without any variant, which makes them unsuitable to assess the mis-splicing effects of variants. Therefore, we excluded them from the analysis. The minigene assay plasmids carrying the ATM gene contexts of two variants (c.3489C>t [在DDP_ATCP_138中]和c.4801a> g [在DDP_ATCP_302中]）没有通过此标准
，因为即使在质粒的ATM基因区域中没有兴趣的变体的情况下，即使在没有兴趣的变体的情况下 ，他们都会显示出利益外显子的主要跳过
。 　　对于C.7865C> t
，总共设计了32个ASO（初始筛选为12个，用于微调筛选20）。ASO被设计为与外显子53中新型剪接供体位点的区域相辅相成 ，或者由C.7865C> t创建的区域
，或者预测外显子53典型剪接供体位点周围的剪接消音器 。这些消音器是根据先前发表的基于己酰胺的Model62预测的。对于C.5763-1050a> g，总共设计了27个ASO（初始筛查为12个 ，进行微调筛查15个）
，与C.5763-1050a> g创建的内含子38中新型剪接供体位点的区域相互补充基于己隔模型）
。对于基于MINIGENE的ASO不合理性的验证，总共为4个ASO无absable变体设计了24个ASO（C.2839-579_2839-576del ，c.2839-581g> a，c.6348-986g> a
，c.6348-986g> t和c.3994-159a> g）。ASO被设计为阻止剪接供体/受体位点或预测感兴趣的伪exon中的外显着剪接消音器 。NT-20和NT-22（具有相同化学性质的非靶向寡核苷酸）用作阴性对照1
。为了进行体外毒性测试，使用了一种具有已知毒性的变形器ASO-TOX。补充表13中提供了所有ASO序列和ASO的详细化学修饰 。所有ASO均通过microsynth制造
。Atipeksen N-1-1临床试验中使用的ASO药物是根据GMP指南制造的。 　　使用Lipofectamine 3000（Thermo Fisher Scientific）将成纤维细胞用200 NM ASO转染 。转染后二十四小时 ，使用PureLink RNA mini（Invitrogen）分离总RNA
。使用寡核-DT和随机六聚体的cDNA合成
，使用Superscript Vilo逆转录酶试剂盒（Invitrogen）进行。对于特定于等位基因的PCR，启动旨在特别排除每个患者的非目标等位基因（扩展数据图6D和9C以及补充表14） 。对于C.5763-1050a> g ，两个ATM变体之间的距离太远（约2 kb）
，无法在琼脂糖凝胶上区分正常和异常剪接的产物（与137 bp）的两个频段
。因此，进行了嵌套的PCR。使用1 µL的cDNA和标准条件（35个循环； 98°C ，5 s
，60°C，持续15 s ，72°C持续45 s）进行PCR 。通过使用ImageJ的1.5％琼脂糖凝胶电泳和光密度分析测量正常剪接转录本的相对数量。 　　如上所述
，将成纤维细胞用400 nm ASO转染
。转染后48小时，使用CAESIUM-137源将细胞用10 Gy辐照 ，然后在37°C下孵育30分钟。然后，使用补充有Roche Phosstop（Sigma-Aldrich）的RIPA缓冲液（波士顿生物产品）收集细胞裂解物 。将裂解物与4x Laemmli缓冲液（Biorad）一起孵育
，并加载到4–15％预制梯度蛋白凝胶（Biorad）上，并通过电泳分离
。然后将蛋白质样品转移到PVDF膜上 ，随后将其与用于磷酸p53的原代抗体（细胞信号技术
，稀释1：500）和磷酸化KAP1（Bethyl Lab，稀释1：1,000）孵育过夜。将GAPDH用作GAPDH（ProteIntech）的一抗载荷对照 ，将其稀释至1：250 。在与磷酸-P53
，磷酸-KAP1和GAPDH（LI-COR）的二级抗体孵育至1：5,000之后，用Li-Cor Odyssey系统可视化靶标 ，并用光密度测定法分析（ImageJ）进行量化
。 　　如上所述
，将成纤维细胞用200 nm的ASO转染。转染后48小时，使用CAESIUM-137源将细胞用1.5 Gy辐照 ，然后在37°C下孵育60分钟 。将细胞在PBS中洗涤
，固定在4％（w/v）多聚甲醛中，并在室温下用0.1％（w/v）Triton X-100透化
。然后将细胞在PBS中孵育过夜 ，该PBS具有3％BSA和抗磷酸-P53（细胞信号技术）和磷酸化-KAP1（贝塔基实验室）的抗体，并用免疫球蛋白G Alexa Fluor共轭物（Life Technologies）可视化。用Hoechst 33342对DNA进行了染色 。与ImageXpress微观显微镜（分子设备）收集图像
，并使用MetaxPress（分子设备）处理
。量化了核中表达的靶标的丰度。 　　使用霓虹灯转染系统（Thermo Fisher Scientific）对成纤维细胞进行电穿孔，其量不同：0-1,000 nm（0、1 、2、5
、5、10 、20
、50、50 、100
、100、200 、500
、1,000 nm;最终浓度） 。电穿孔后二十四小时 ，如上所述分离总RNA。如上所述
，进行了cDNA合成，RT -PCR ，凝胶电泳和光密度法
。 　　如上所述
，将成纤维细胞用200 nm的ASO转染。转染后48小时，如上所述分离总RNA 。使用KAPA Hyper Prep Kit（KAPA生物系统）制备RNA-Seq库
。测序是在Illumina Hiseq 2500上进行的（用于测序； 2×100 bp）。为了对齐 ，使用Star（v.2.7.5c）（参考文献63）在配对端的两通道模式中绘制grch38/hg38上的读数
，以产生由染色体坐标排序的BAM文件 。未向对齐程序提供基因注释，以避免有利于注释的剪接连接的有偏见的对齐
。使用SamTools（V.1.10）（参考文献64）对排序的BAM文件进行索引。IGV用于绘制生鱼片图 ，该图显示了支撑剪接连接的读取数量 。 　　以下衍生序列是从AT008（Atipeksen）
，AT026，AT056 ，Nusinersen和Milasen的AT008序列（ATIPEKSEN）和MILASEN的计算产生的：（1）具有逐渐修剪末端的序列，从全长ASO序列开始
，长度降低到长度为16 nt的序列；（2）最多2 NT不匹配的序列；（3）具有1-NT内部插入或缺失的序列（补充图5–8和11）
。BWA（V.0.7.17）（参考文献46）用于对齐GRCH38/HG38上的生成序列，并从UCSC基因组浏览器下载了RefSeq Transcriptome序列。 　　FITC膜联蛋白V凋亡检测试剂盒I（BD 556547 ，BD Biosciences）用于定量测量以上所述
，在不同浓度的ASOS转染后经历凋亡后经历凋亡的细胞百分比 。收集细胞，用PBS洗涤 ，并在转染后四天重悬于1×结合缓冲液中
。用5 µL膜联蛋白V-FITC和5 µL碘化丙啶（PI）在室温下在室温下用5 µL膜联蛋白V-FITC和5 µL碘化丙啶（PI）染色了500微米的重悬细胞。使用流式细胞仪（BD FACSARIA III系统）分析细胞
，并通过FlowJo软件进行量化 。膜联蛋白-V阳性和PI阴性分数是“早期凋亡”，Annexin-V阳性和PI阳性的分数为“晚期凋亡或坏死性
”。 　　有关研究设计的更多信息可在与本文有关的自然投资组合报告摘要中获得
。