Searching for somatic mutations in McCune-Albright syndrome: a comparative study of the peptidic nucleic acid versus the nested PCR method based on 148 DNA samples
Searching for somatic mutations in McCune-Albright syndrome: a comparative study of the peptidic nucleic acid versus the nested PCR method based on 148 DNA samples
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DOI：
10.1530/eje.1.02301
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发表时间：
2006-12-01
影响因子：
5.8
通讯作者：
Sultan, C.
中科院分区：
文献类型：
--
作者：
Kalfa, N.;Philibert, P.;Sultan, C.
背景：已在mcune - albright综合征（MAS）患者中发现编码刺激G蛋白a亚基的Gs α基因（GNAS）的激活突变。MAS分子诊断的准确性和敏感性对于最佳治疗策略和适应的随访是必需的，特别是对于不完整的MAS临床形式。迄今为止，高度敏感的巢式PCR方法在突变位点通过限制性内切酶进行中间酶切是最广泛使用的技术之一。本研究评价了一种新的多肽核酸（PNA）诊断方法，并与巢式PCR方法进行了比较。材料和方法：纳入88例临床症状符合MAS的患者的148份DNA样本。DNA样本主要取自外周血、卵巢组织或囊肿液、骨病变。巢式PCR法需要4天。PNA夹紧需要1.5天，利用更高的热稳定性和PNA- dna偶联的特异性来抑制PCR产物的形成。随后对所有病例进行直接测序。结果：巢式PCR检测突变的灵敏度为54% (n=80)， PNA检测突变的灵敏度为46.6% (n=69) （P < 0.05）。PNA未检测到突变的11例主要是不完全和不典型的MAS临床形式（n = 10/11）。每个样品的PNA夹夹成本为50欧元，而巢式PCR为136欧元。结论：PNA夹持是诊断MAS快速、可靠、经济的方法。它应该是一线诊断方法，尽管阴性结果，特别是对于不完整的MAS临床形式，应通过巢式PCR确认。
Background: Activating mutations of the Gs alpha gene (GNAS), which encodes for the a-subunit of the stimulatory G protein, have been identified in patients with McCune-Albright syndrome (MAS). Accuracy and sensitivity in the molecular diagnosis of MAS is mandatory for optimal therapeutic strategy and adapted follow-up, especially for incomplete clinical forms of MAS. To date, the highly sensitive nested PCR method with intermediary digestion by a restriction enzyme at the mutation site is one of the most widely used techniques. This study evaluated a new diagnostic method using a peptidic nucleic acid (PNA) and compared it with the nested PCR method.Material and methods: One hundred and forty-eight DNA samples from eighty-eight patients presenting clinical symptoms compatible with MAS were included. The DNA samples were mainly obtained from peripheral blood, ovarian tissue or cyst liquid, and bone lesions. The nested PCR method required 4 days. PNA clamping required 1.5 days and utilized the higher thermal stability and specificity of PNA-DNA coupling to inhibit PCR product formation. Direct sequencing was subsequently performed in all cases.Results: The sensitivity of mutation detection was 54% (n=80) for nested PCR and 46.6% (n=69) for PNA (P > 0.05). The 11 cases where PNA failed to detect the mutation were mainly incomplete and atypical clinical forms of MAS (n = 10/11). The cost per sample was 50 euros for PNA clamping versus 136 euros for nested PCR.Conclusion: PNA clamping is a rapid, reliable, and economical method to diagnose MAS. It should be the first-line diagnostic method, although negative results, especially for incomplete clinical forms of MAS, should be confirmed by nested PCR.