PDF(Pubmed)
Abstract：
Genetic variants in ATP7A are associated with a spectrum of X-linked disorders. In descending order of severity, these are Menkes disease, occipital horn syndrome, and X-linked distal spinal muscular atrophy. After 30 years of diagnostic investigation, we identified a deep intronic ATP7A variant in four males from a family affected to variable degrees by a predominantly skeletal phenotype, featuring bowing of long bones, elbow joints with restricted mobility which dislocate frequently, coarse curly hair, chronic diarrhoea, and motor coordination difficulties. Analysis of whole genome sequencing data from the Genomics England 100,000 Genomes Project following clinical re-evaluation identified a deep intronic ATP7A variant, which was predicted by SpliceAI to have a modest splicing effect. Using a mini-gene splicing assay, we determined that the intronic variant results in aberrant splicing. Sanger sequencing of patient cDNA revealed ATP7A transcripts with exon 5 skipping, or inclusion of a novel intron 4 pseudoexon. In both instances, frameshift leading to premature termination are predicted. Quantification of ATP7A mRNA transcripts using a qPCR assay indicated that the majority of transcripts (86.1 %) have non-canonical splicing, with 68.0 % featuring exon 5 skipping, and 18.1 % featuring the novel pseudoexon. We suggest that the variability of the phenotypes within the affected males results from the stochastic effects of splicing. This deep intronic variant, resulting in aberrant ATP7A splicing, expands the understanding of intronic variation on the ATP7A-related disease spectrum.
摘要：
ATP7A的遗传变异与一系列X连锁疾病有关。按照严重性的降序，这些是门克斯病，枕角综合征,和X连锁远端脊髓性肌萎缩症。经过30年的诊断调查，我们在一个主要受骨骼表型影响程度不同的家族中的四名男性中发现了一个深内含子ATP7A变体，以弯曲长骨为特征，活动受限的肘关节经常脱臼，粗糙的卷发，慢性腹泻,和运动协调困难。在临床重新评估后，对来自GenomicsEngland100,000基因组项目的全基因组测序数据进行分析，确定了一个深内含子ATP7A变体，SpliceAI预测具有适度的剪接效果。使用微型基因剪接测定法，我们确定内含子变体导致异常剪接。患者cDNA的Sanger测序显示ATP7A转录本具有外显子5跳跃，或包含一个新的内含子4伪外显子。在这两种情况下，预测会导致过早终止的移码。使用qPCR测定法对ATP7AmRNA转录本进行定量表明，大多数转录本（86.1％）具有非规范剪接，68.0%有外显子5跳跃，18.1％的人以小说伪外显子为特色。我们建议受影响的男性中表型的变异性是剪接的随机效应所致。这种深内含子变体，导致ATP7A剪接异常，扩展了对ATP7A相关疾病谱内含子变异的理解。
