Data Sheet 1_Genetic and in silico functional characterization of a novel structural variant in the PAH gene by long-reads sequencing and structural modeling.docx

IntroductionPhenylketonuria (PKU) is an inherited metabolic disorder caused by biallelic variants in the PAH gene, leading to phenylalanine accumulation and progressive neuronal damage. Over 3,000 variants have been described worldwide; however, a previously unreported exon duplication was identified in Chile, whose genetic and functional characteristics remained unknown. MethodsA patient carrying a duplication of exon 2 in the PAH gene, previously detected by MLPA, was analyzed using nanopore sequencing coupled with CRISPR/Cas9 enrichment (nCATS) to determine the location, size, and orientation of the variant. Specific fragment amplification by PCR and Sanger sequencing was subsequently performed on samples from this patient and seven additional individuals to confirm the presence of the structural variant. Structural modelling of the resulting PAH protein was also conducted to predict functional consequences. ResultsThe nCATS technique identified a ∼18 kb tandem duplication between exons 1 and 3 of the PAH gene. This exon duplication was confirmed by PCR and Sanger sequencing in all eight patients. Additionally, an adenine insertion was detected at the junction site of the duplication. Structural modelling predicted an additional N-terminal segment that would likely interfere with sensing of phenylalanine. DiscussionThe clinical, genetic and in silico functional characterization of this variant, using nCATS and structural modeling, suggests a mild, but relevant alteration in PAH enzymatic function. These findings support the delineation of genotype-phenotype correlations for complex structural variants, which may contribute to the development of personalized therapeutic strategies, while enriching both national and international PKU variant databases.引言：苯丙酮尿症（Phenylketonuria, PKU）是一种由PAH基因双等位基因变异引发的遗传性代谢紊乱，可导致苯丙氨酸蓄积并引发进行性神经元损伤。目前全球已报道超过3000种该基因变异，但此前在智利人群中发现了一种未被报道过的外显子重复变异，其遗传与功能特征尚未明确。 方法：本研究对1例经多重连接依赖探针扩增（Multiplex Ligation-dependent Probe Amplification, MLPA）检测出PAH基因2号外显子重复的患者，采用纳米孔测序联合CRISPR/Cas9富集技术（nanopore sequencing coupled with CRISPR/Cas9 enrichment, nCATS）进行分析，以明确该变异的位置、片段大小与方向。随后通过聚合酶链式反应（Polymerase Chain Reaction, PCR）特异性扩增该目标区域，并对该患者及另外7名受试者的样本进行桑格测序（Sanger sequencing），以验证该结构变异的存在。同时还对编码产生的PAH蛋白进行结构建模，以预测其功能影响。 结果：nCATS技术明确了PAH基因1号与3号外显子之间存在一段约18kb的串联重复序列。经PCR与桑格测序验证，该外显子重复变异在全部8名受试者中均存在。此外，在该重复序列的连接位点处检测到1个腺嘌呤插入突变。结构建模预测，该变异会新增一段N端片段，极有可能干扰苯丙氨酸的感知过程。 讨论：本研究通过nCATS技术与结构建模，完成了该变异的临床、遗传与计算机模拟功能表征，结果提示PAH的酶功能存在轻度但具有临床意义的改变。上述发现有助于明确复杂结构变异的基因型-表型关联，可为个性化治疗策略的开发提供支撑，同时也将丰富全球及智利本国的PKU变异数据库。