Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells
Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells
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DOI：
10.1161/circulationaha.117.032273
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发表时间：
2018-12-04
期刊：
影响因子：
37.8
通讯作者：
Wu, Joseph C.
中科院分区：
文献类型：
--
作者：
Ma, Ning;Zhang, Joe Z.;Wu, Joseph C.
工作背景：向低成本和快速的下一代测序的进展已经发现了患者和无症状健康个体中的大量不确定意义（VUS）的变体。VUS是一种罕见或新型变异，其疾病致病性尚未得到最终证明或排除，因此无法明确注释。因此，VUS提出了关键的临床解释和风险评估挑战，迫切需要新的方法来更好地表征其致病性。方法：为了解决这一挑战并展示基因组变异解释的不确定性，我们招募了一名健康的无症状个体，缺乏心脏病临床病史，携带肥厚性心肌病（HCM）相关遗传变异170 C>A，NP_000249.1：p.Ala57Asp），ClinVar数据库报道可能是致病性的。人诱导多能干细胞（iPSC）来源于杂合VUSMYL 3（（170 C>A））载体，并且使用CRISPR/Cas9编辑其基因组以产生4个等基因iPSC系：（1）校正的健康对照;（2）纯合VUSMYL 3（（170 C>A））;（3）杂合移码突变MYL 3（170 C>A）。（（170 C>A/fs））;和（4）已知的杂合MML 3致病突变（NM_000258.2：c.170C>G），与VUSMML 3（（170 C>A））系的核苷酸位置相同。对同基因iPSC衍生的心肌细胞（iPSC-CMs）进行广泛的测定，包括基因表达、肌节结构、细胞大小、收缩性、动作电位和钙处理的测量。结果：杂合VUSMYL 3（（170 C>A））-iPSC-CMs在基因表达、形态学或功能水平上未显示HCM表型。此外，基因组编辑的纯合VUSMYL 3（（170 C>A））-和移码突变MYL 3（（170 C>A/fs））-iPSC-CM系也是无症状的，支持对该特定MYL 3变体的良性评估。对携带已知致病性MYL 3突变MYL 3（（170 C>G））的基因组编辑的等基因系和携带MYBPC 3（（961 G>A））HCM变体的载体特异性iPSC-CM系的致病性的进一步评估证明了该组合平台提供致病性和良性评估的能力。我们的研究说明了载体特异性iPSC的成簇的规则间隔的短回文重复序列/Cas9基因组编辑在培养皿中以载体特异性方式阐明良性和致病性HCM功能表型的能力。因此，该平台代表了一种很有前途的VUS风险评估工具，可用于评估HCM相关的VUS，以及一般的VUS，从而为这一新兴领域的精确医学工具库做出了重大贡献。
Background: The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic healthy individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity.Methods: To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a healthy asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be likely pathogenic. Human-induced pluripotent stem cells (iPSCs) were derived from the heterozygous VUSMYL3((170C>A)) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected healthy control; (2) homozygous VUSMYL3((170C>A)); (3) heterozygous frameshift mutation MYL3((170C>A/fs)); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUSMYL3((170C>A)), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs).Results: The heterozygous VUSMYL3((170C>A))-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUSMYL3((170C>A))- and frameshift mutation MYL3((170C>A/fs))-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3((170C>G)), and a carrier-specific iPSC-CMs line, carrying a MYBPC3((961G>A)) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments.Conclusions: Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrier-specific manner in a dish. As such, this platform represents a promising VUS risk-assessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.