a Institute of Laboratory Medicine, Triemli Hospital , Zurich , Switzerland.
b Center for Cardiovascular Genetics and Gene Diagnostics, Foundation for People with Rare Diseases , Schlieren-Zurich , Switzerland.
Scand J Clin Lab Invest. 2019 Sep;79(5):305-313. doi: 10.1080/00365513.2019.1622030. Epub 2019 Jun 1.
Molecular diagnosis of autosomal dominant acute hepatic porphyrias (AHPs) plays an important role in the management of these disorders. To introduce next generation sequencing (NGS) to the porphyria diagnosis, we designed a panel that contained four genes, , and for mutational analysis of acute intermittent porphyria (AIP), hereditary coproporphyria (HCP) and variegate porphyria (VP). To validate the AHP panel, 30 samples with known pathogenic variants as determined by Sanger sequencing, were analyzed using the Ion PGM™. Among them, nine have so far not been reported. The pathogenic variants were identified and annotated manually in IGV by three individuals who were blinded to the Sanger results. The AHP panel consists of 95 amplicons that covers 92% of the coding region of the four genes. Of the 95 amplicons, 93 had an average read-depth of >500 reads. In 29 of the 30 tested samples, pathogenic variants were correctly identified and annotated. The number of reads from the mutated alleles were approximately 50% of the total. The annotation of a 22-bp duplication with NGS differed from that of Sanger by one nucleotide. NGS showed an advantage in allelic discrimination over Sanger sequencing and was also able to detect a known somatic variant in the gene. The AHP panel will be applied in the initial diagnosis of new patients. Any sequence variations with a frequency of ≥10% will be confirmed by Sanger sequencing. The cost-effectiveness of a NGS approach for AHP in a diagnostic laboratory needs to be further assessed.
常染色体显性遗传急性肝性卟啉症（AHP）的分子诊断在这些疾病的管理中起着重要作用。为了将下一代测序（NGS）引入卟啉症诊断，我们设计了一个包含四个基因、、和的panel，用于突变分析急性间歇性卟啉症（AIP）、遗传性粪卟啉症（HCP）和变异性卟啉症（VP）。为了验证 AHP panel，我们使用 Ion PGM™ 对 30 个经 Sanger 测序确定具有已知致病性变异的样本进行了分析。其中，有 9 个目前尚未报道。这 30 个样本的致病性变异是由三个人手动在 IGV 中识别和注释的，这三个人并不知道 Sanger 结果。AHP panel 由 95 个扩增子组成，覆盖了四个基因的 92%编码区。在 95 个扩增子中，93 个的平均读取深度大于 500 个reads。在 30 个测试样本中，有 29 个正确识别和注释了致病性变异。突变等位基因的reads 数约为总reads 的 50%。NGS 对 22-bp 重复的注释与 Sanger 测序的结果不同，仅相差一个核苷酸。NGS 在等位基因检测方面优于 Sanger 测序，还能够检测到基因中的一个已知体细胞变异。AHP panel 将应用于新患者的初步诊断。任何频率≥10%的序列变异都将通过 Sanger 测序进行确认。在诊断实验室中，NGS 方法用于 AHP 的成本效益需要进一步评估。
