A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11.2-12 duplications and deletions leading to HMSN/HNPP
A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11.2-12 duplications and deletions leading to HMSN/HNPP
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DOI：
10.1038/sj.ejhg.5200920
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发表时间：
2003-02-01
影响因子：
5.2
通讯作者：
Reis, A
中科院分区：
文献类型：
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作者：
Thiel, CT;Kraus, C;Reis, A
染色体17p11.2-12上的1.4Mb串联重复，包括外周髓鞘蛋白22（PMP 22）基因，导致70%的Charcot-Marie-Tooth病或遗传性运动和感觉神经病I（CMT 1A/HMSN I）脱髓鞘1型病例。这个CMT 1A区域的相互缺失导致易患压力性麻痹的遗传性神经病（HNPP）。CMT 1A重复使PMP 22基因剂量从2增加到3，HNPP缺失使基因剂量从2减少到1。目前，HMSN/HNPP患者的常规诊断主要用CMT 1A区域侧翼重复元件之间的多态性标记进行。这些在CMT 1A重复的情况下显示定量和/或定性变化，在HNPP缺失的情况下显示纯合等位基因模式。在HNPP患者中，缺失通常通过荧光原位杂交（FISH）确认。我们建立了一种可靠的单管实时定量PCR方法，可直接快速测定PMP 22基因的剂量。该方法涉及使用FAM标记的Taqman探针和VIC标记的探针的多重反应，所述Taqman探针具有来自PMP 22外显子3的塔姆拉猝灭剂，所述VIC标记的探针具有来自白蛋白基因的外显子12的非荧光猝灭剂作为内参。通过比较阈值循环法（DeltaDeltaCt）确定PMP 22基因的拷贝数。每个样品以四倍运行，并在两个不同的阈值水平下进行分析。对给出最小标准差的水平进行评分。我们通过对252例已知基因型的HMSN患者的回顾性分析评估了这种方法，并在99%的病例中证实了先前的发现。两名患者被错误诊断与微卫星分析，而定量实时PCR确定了正确的基因型，证实了FISH。因此，该方法显示出对微卫星分析的上级灵敏度，并且具有作为用于定量分析CMT 1A和HNPP的快速且均匀的测定的额外优点。
A 1.4-Mb tandem duplication, including the gene for peripheral myelin protein 22 (PMP22) in chromosome 17p11.2-12 is responsible for 70% of the cases of the demyelinating type 1 of Charcot-Marie-Tooth disease or hereditary motor and sensory neuropathy I (CMT1A/HMSN I). A reciprocal deletion of this CMT1A region causes the hereditary neuropathy with liability to pressure palsies (HNPP). The CMT1A duplication increases the PMP22 gene dosage from two to three, the HNPP deletion reduces the gene dosage from two to one. Currently, routine diagnosis of HMSN/HNPP patients is mainly performed with polymorphic markers in-between the repetitive elements flanking the CMT1A region. These show quantitative and/or qualitative changes in case of a CMT1A duplication and a homozygous allele pattern in case of HNPP deletion. In HNPP patients the deletion is usually confirmed by fluorescence in situ hybridisation (FISH). We now developed a reliable, single tube real-time quantitative PCR assay for rapid determination of PMP22 gene dosage directly. This method involves a multiplex reaction using FAM labelled, Taqman-probe with TAMRA quencher derived from PMP22 exon 3 and a VIC labelled probe with non-fluorescent quencher from exon 12 of the albumin gene as internal reference. Copy number of the PMP22 gene was determined by the comparative threshold cycle method (DeltaDeltaCt). Each sample was run in quadruplicate and analysed at two different threshold levels. The level giving the smallest standard deviation was scored. We evaluated this method through the retrospective analysis of 252 HMSN patients with known genotype and could confirm the previous findings in 99% of cases. Two patients were wrongly diagnosed with microsatellite analysis while quantitative real-time PCR identified the correct genotype, as confirmed by FISH. Thus, this method shows superior sensitivity to microsatellite analysis and has the additional advantage of being a fast and uniform assay for quantitative analysis of both CMT1A and HNPP.