Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA; Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15261, USA.
Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, and UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA 15224, USA.
Mol Genet Metab. 2021 Sep-Oct;134(1-2):29-36. doi: 10.1016/j.ymgme.2021.08.012. Epub 2021 Aug 30.
Clinical standard of care for newborn screening (NBS) is acylcarnitine metabolites quantitation by tandem mass spectrometry (MS/MS) from dried blood spots. Follow up sequencing often results in identification of one or more variants of uncertain significance (VUS). Isovaleric acidemia (IVA) is an autosomal recessive inborn error of metabolism caused by deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the Leu catabolism pathway. Many IVD mutations are characterized as VUS complicating IVA clinical diagnoses and treatment. We present a testing platform approach to confirm the functional implication of VUS identified in newborns with IVA applicable to multiple inborn errors of metabolism identified by NBS.
An IVD null HEK293T cell culture model was generated by using a dual sgRNA CRISPR/Cas9 genome-editing strategy targeting IVD exons 2-3. Clonal cell lines were confirmed by a combination of genomic breakpoint sequencing and droplet digital PCR. The IVD null model had no IVDH antigen signal and 96% reduction in IVDH enzyme activity. The IVD null model was transfected with vectors containing control or variant IVD and functional assays were performed to determine variant pathogenicity.
c.149G > C (p.Arg50Pro; precursor numbering), c.986T > C (p.Met329Thr), and c.1010G > A (p.Arg337Gln), c.1179del394 f. mutant proteins had reduced IVDH protein and activity. c.932C > T (p.Ala311Val), c.707C > T (p.Thr236Ile), and c.1232G > A (p.Arg411Gln) had stable IVDH protein, but no enzyme activity. c.521T > G (p.Val174Gly) had normal IVDH protein and activity. IVD variant transfection results confirmed results from IVA fibroblasts containing the same variants.
We have developed an IVD null HEK293T cell line to rapidly allow determination of VUS pathogenicity following identification of novel alleles by clinical sequencing following positive NBS results for suspected IVA. We suggest similar models can be generated via genome-editing for high throughput assessment of VUS function for a multitude of inborn errors of metabolism and can ideally supplement NBS programs.
新生儿筛查（NBS）的临床标准护理是通过串联质谱（MS/MS）定量分析干血斑中的酰基肉碱代谢物。后续测序通常会确定一个或多个意义不明的变异（VUS）。异戊酸血症（IVA）是一种常染色体隐性遗传的先天性代谢缺陷，由亮氨酸代谢途径中的异戊酰辅酶 A 脱氢酶（IVDH）缺乏引起。许多 IVD 突变被认为是 VUS，这使得 IVA 的临床诊断和治疗变得复杂。我们提出了一种测试平台方法，以确认通过 NBS 鉴定的患有 IVA 的新生儿中鉴定出的 VUS 的功能意义，该方法适用于多种代谢性疾病。
使用靶向 IVD 外显子 2-3 的双 sgRNA CRISPR/Cas9 基因组编辑策略，生成 IVD 无效的 HEK293T 细胞培养模型。通过基因组断点测序和液滴数字 PCR 的组合来确认克隆细胞系。IVD 无效模型没有 IVDH 抗原信号，IVDH 酶活性降低 96%。将 IVD 无效模型转染含有对照或变体 IVD 的载体，并进行功能测定以确定变体的致病性。
c.149G>C（p.Arg50Pro；前体编号）、c.986T>C（p.Met329Thr）和 c.1010G>A（p.Arg337Gln）、c.1179del394f 突变蛋白的 IVDH 蛋白和活性降低。c.932C>T（p.Ala311Val）、c.707C>T（p.Thr236Ile）和 c.1232G>A（p.Arg411Gln）具有稳定的 IVDH 蛋白，但没有酶活性。c.521T>G（p.Val174Gly）具有正常的 IVDH 蛋白和活性。IVA 成纤维细胞中含有相同变体的 IVD 变体转染结果证实了这一结果。
我们已经开发了一种 IVD 无效的 HEK293T 细胞系，以便在通过临床测序鉴定出疑似 IVA 的 NBS 阳性结果后的新型等位基因后，快速确定 VUS 的致病性。我们建议可以通过基因组编辑生成类似的模型，用于高通量评估多种先天性代谢缺陷的 VUS 功能，并可理想地补充 NBS 计划。
