探討新穎C19orf12致病基因突變導致顯性遺傳腦部鐵質沉積神經退化症之機轉

Abstract

研究背景與目的 腦部鐵質沉積神經退化症 (Neurodegeneration with Brain Iron Accumulation, NBIA)，為少見之遺傳性神經退化性疾病，臨床症狀漸進式肢體出現肌張力不全症、巴金森緩慢僵硬症狀、痙攣與合併認知功能下降等多重神經系統退化症狀。腦部核磁共振掃描可於基底核以及腦幹黑質區發現深色鐵質沉澱的影像特徵，腦部病理報告則於這些區域觀察到局部萎縮、神經細胞中深色鐵質沉積、以a-synuclein為主成分的路易士體或是tau病理蛋白沉積。NBIA具有10多種分型，各分型為不同基因的突變導致體顯性或是隱性之遺傳模式。隨著分子生物學的進展，迄今為止已知至少10種基因參與鐵質在細胞中之運輸、儲存和調控與神經細胞功能的維持、脂質代謝與粒線體功能息息相關。然而，這些基因的突變與各基因之間的關聯交互作用如何導致神經細胞中病理蛋白沉積、鐵質代謝異常因而鐵質沉積與神經細胞凋亡機轉仍未知。 我們過去發現一少見之顯性遺傳NBIA家族，並不帶有已知肌張力不全症、巴金森症之遺傳基因，病患與母親皆於20-30歲間出現漸進式肌張力不全症、僵硬與合併認知功能下降等多重神經系統退化症狀，腦部核磁共振掃描亦出現典型之鐵質沉澱的影像特徵。因此，我們希望：一、透過全外顯子定序技術(whole exome sequencing, WES)找出此顯性遺傳家族之新穎致病基因變異。二：利用CRISPR/Cas9 基因編輯技術建立帶有此新穎致病基因變異神經細胞模式，以探討神經細胞退化之分子機轉，以期將來為NBIA神經退行性疾病的研究和治療提供策略。 研究結果 此顯性遺傳NBIA家族中兩位罹病成員接受WES分析，在層層分析，僅挑選在公開基因資訊網站gnomAD與Taiwan biobank中minor allele frequency 小於0.0001的基因變異位點，並經PolyPhen-2 與SIFT programs預測可能會產生具致病性蛋白質的變異後，我們發現在該家族罹病成員中的C19orf12基因上有一異型合子(heterozygous)的單一核甘酸嵌入變異c.273_274insA ，此變異導致胺基酸提前終止合成，因而造成錯位突變(p.P92Tfs*9)，家族成員內的分離分析(Segregation analysis)亦顯示此嵌入錯位突變應為此顯性家族之致病位點。值得注意的是C19orf12 為導致NBIA分型中少見之mitochondrial membrane protein-associated neurodegeneration (MPAN)之致病基因，C19orf12 轉錄之蛋白為表現在粒線體與內質網之17kDa蛋白，相關研究極少，我們利用CRISPR/Cas9 基因編輯技術建立帶有此新穎致病基因變異的神經細胞模式，以探討此突變導致神經細胞退化之分子機轉，並藉由探討粒線體功能、脂質代謝與鐵質代謝異常之交互關係，以期釐清此少見之神經退化性疾病的分子生理機轉，希望有助於未來發展此疾病之治療策略。

Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of rare inherited neurological disorders that share the clinical features of dystonia, parkinsonism, spasticity accompanied by varying degrees of intellectual disability and abnormal iron deposition in the basal ganglia and/or substantia nigra. Iron accumulates in the basal ganglia and may be accompanied by a-synuclein containing Lewy bodies deposition, axonal swellings and hyperphosphorylated tau depending on NBIA subtype. More than 10 genes have been associated with different subtypes of NBIA contributing to either autosomal-dominant or autosomal recessive inheritances of disease. However, the molecular mechanism and the interactions between these NBIA-causative genes in neurodegeneration, iron accumulations remain largely unknown. We previously identified an autosomal-dominant NBIA family with characteristics clinical and radiological features through large-scale screening of patients with dystonia. We therefore have the following aims to identify the genetic cause and the molecular mechanism leading to neurodegeneration in this index family. Aim 1: Utilizing whole exome sequencing (WES) and segregation analysis to identify the genetic cause in the autosomal-dominant NBIA family. Aim 2: To investigate the pathogenicity of the identified mutation using clustered, regularly interspaced, short palindromic repeats-associated nuclease 9 (CRISPR-Cas9)-based knock-in human dopaminergic SH-SY5Y cell line model. Results: WES was performed in two affected members of this autosomal-dominant inheritance NBIA family with a coverage of 100× read depth showed 96 nonsynonymous variants with minor allele frequency ≤ 0.0001 in the gnomAD and Taiwan Biobank. Further comparative analyses and pathogenicity prediction analysis by PolyPhen-2 and SIFT programs identified one heterozygous novel and potentially pathogenic variant, c.273_274insA (p.P92Tfs*9) insertion in C19orf12, which caused a frameshift and premature stop codon in the protein sequence. This novel potential pathogenic variant co-segregated within the family. Mutations in C19orf12 contribute to a rare subtype of NBIA, mitochondrial membrane protein-associated neurodegeneration (MPAN). To prove its pathogenicity and functional impacts in the neurodegeneration, we will assay the pathogenicity of the identified mutation using CRISPR/Cas9-based knock-in human dopaminergic SH-SY5Y cell line model. Further neuronal iron dynamic assay, mitochondria function and fission-fusion dynamics and protein degradation pathways will be investigated to prove the pathogenicity of this novel mutation of MPAN. Conclusion: Our findings will support recent observations that monoallelic C19orf12 mutations may contribute to autosomal dominant MPAN and will be the first report showing the pathogenic mechanism of C19orf12 mutation in neurons.