脊髓性肌萎缩症核酸飞行质谱基因检测的方法建立及临床应用研究

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterised by loss of motor neurons and progressive muscle wasting. SMA is one of the main genetic diseases leading to neonatal and child death, with an incidence of 1 in 6000 to 10000 newborns. The carrier frequency in Chinese is 1 in 42. .Genetic mutations in SMN1 are the main cause for SMA. Among newborns with SMA, homozygous deletion of SMN1 accounts for 95%, while compound heterozygous mutations (with one SMN1 allele deleted and the other SMN1 allele containing a point mutation) account for 5%. The highly homologous SMN2 codes for 10-20% functional proteins. SMN2 copy number increase may be partially compensative, leading to milder symptoms. Thus, accurate SMA genetic testing and carrier screening demands a comprehensive analysis of SMN1 and SMN2 copy numbers, SMN1 disease-causing point mutations and other relevant genes (e.g. NAIP). Due to the high homology, the analysis of SMN1 is often complicated by SMN2. .Common methods for SMA genetic testing include MLPA, qPCR and DNA sequencing. Although each method has its merits, none of these methods can achieve the comprehensiveness (for both quantitative and qualitative analysis) required by the genetic complexity of the disease. We propose to use MALDI-TOF mass spectrometry, combined with real competitive PCR and multiplex point mutation detection, to achieve a highly sensitive and accurate DNA testing of both SMA patients and carriers. In addition, we propose to evaluate the clinical utility of this method for prenatal testing of SMA.

脊髓性肌萎缩症（SMA）是一种常染色体隐性遗传的神经退行性疾病，是婴幼儿致死的主要遗传疾病之一。SMA的携带者则高达1/42。.SMN1基因突变是最主要的SMA致病原因。SMA患儿中，SMN1纯合缺失占95%，复合杂合突变（杂合缺失伴点突变）占5%。高度同源的SMN2仅表达少量功能性蛋白，其拷贝数扩增在SMA病人中可以起到部分补偿作用。所以，SMA患儿的基因诊断和携带者的筛查，需要对SMN1、SMN2的拷贝数、SMN1多个点突变、及其它相关基因进行同时检测。.SMA基因检测的主要方法有MLPA、qPCR、测序等，虽然各有优点，但是都无法实现如上所述的全面定量和定性多重检测。本申请拟采用核酸飞行质谱方法，结合real competitive PCR和点突变高度多重检测技术，实现对绝大部分携带者和患者的精确基因检测，并且验证此方法在SMA产前检测中的临床应用价值。

本研究基于核酸飞行质谱平台建立了脊髓性肌萎缩症基因诊断的检测方案，可以同时检测SMN1和SMN2拷贝数和19种较常见的SMN1点突变。.其一为结合real-competitive PCR技术建立的SMA致病基因SMN1拷贝数的检测、及其修饰基因SMN2基因和NAIP基因拷贝数的检测的SMA疾病相关基因拷贝数定量方案。我们应用该方案在先后两组独立的临床样本中进行了检测，同时应用MLPA技术（金标准）做了平行检测用于该方案的性能评估。.其二为依据国内外研究报道汇总了19种较常见的SMN1点突变，建立了基于核酸飞行质谱平台的单管检测19种常见SMN1基因点突变的检测方案。该检测方案的有效性也得到了初步的验证。.本研究建立的SMA疾病相关基因拷贝数定量可准确地诊断SMA患者和筛查SMA携带者（SMN1基因缺失型）。SMN1基因点突变的检测可以进一步提升SMA患者和携带者的检出率。核酸飞行质谱平台具有自动化、高通量的特点，易于在临床展开大规模、准确且高效的新生儿SMA筛查及在特定人群(比如年轻代孕夫妇)中的SMA携带者筛查。.此外，本研究进一步拓展了基于高通量测序技术平台的SMA检测方案，可准去地对SMN1和SMN2进行定量。通过建库方案、捕获方案、生信分析的优化，该方案有望进一步应用于SMA的无创产前检测。