技术进步在DNA改变鉴定中的临床应用一直导致遗传医学诊断产量的提高。在染色体一侧,从细胞遗传学技术评估数量和总体结构缺陷到基因组微阵列检测隐蔽拷贝数变异,在分子水平上,从Sanger方法研究单基因的核苷酸序列到高通量下一代测序(NGS)技术,分辨率和灵敏度逐渐增加,可检测的DNA异常和已知遗传原因的孟德尔疾病的范围大大扩大。然而,特定的基因组区域(即,重复和富含GC的序列)通过标准基因测试进行低效分析,仍然依靠费力,耗时且灵敏度低的方法(即,用于重复扩增的Southern印迹或具有高度同源假基因的基因的长PCR),至少部分未确诊的遗传性疾病患者。第三代测序,生成具有改进的Mappability的长读取,更适合于检测难以接近的基因组区域中的结构改变和缺陷。虽然最近实施,但尚未临床应用,长读测序(LRS)技术已经显示出其在遗传医学研究中的潜力,这可能会极大地影响诊断产量和报告时间,通过将它们转化为临床环境。主要研究的LRS应用涉及结构变体和重复扩展的识别,可能是因为它们的检测技术没有那些致力于单核苷酸变异(SNV)鉴定的技术发展得那么快:金标准分析是核型分析和平衡和不平衡染色体重排的微阵列,分别,以及Southern印迹和重复引物PCR,用于扩增和确定扩增等位基因的大小,受到有限的分辨率和灵敏度的损害,而NGS的出现并未显着改善。然而,最近,随着最新产品版本提供的更高的准确性,LRS也已经过SNV检测测试,特别是在具有高度同源假基因的基因中,以及用于单倍型重建以评估具有从头致病变异的等位基因的亲本起源。我们提供了有关最新科学论文的综述,这些论文探讨了LRS在遗传疾病诊断中的潜力及其在常规基因检测中的潜在未来应用。
The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.