Thiel Christian T, Kraus Cornelia, Rauch Anita, Ekici Arif B, Rautenstrauss Bernd, Reis André
Institute of Human Genetics, University of Erlangen-Nuremberg, Schwabachanlage 10, 91054 Erlangen, Germany.
Eur J Hum Genet. 2003 Feb;11(2):170-8. doi: 10.1038/sj.ejhg.5200920.
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.
17号染色体p11.2 - 12区域的一个1.4兆碱基对的串联重复，包括外周髓磷脂蛋白22（PMP22）基因，是导致70%的1型脱髓鞘性夏科 - 马里 - 图斯病或遗传性运动和感觉神经病I（CMT1A/HMSN I）病例的原因。该CMT1A区域的相互缺失导致易患压迫性麻痹的遗传性神经病（HNPP）。CMT1A重复使PMP22基因剂量从两个增加到三个，HNPP缺失使基因剂量从两个减少到一个。目前，HMSN/HNPP患者的常规诊断主要通过CMT1A区域两侧重复元件之间的多态性标记进行。在CMT1A重复的情况下，这些标记显示出数量和/或质量上的变化，在HNPP缺失的情况下显示出纯合等位基因模式。在HNPP患者中，缺失通常通过荧光原位杂交（FISH）来确认。我们现在开发了一种可靠的单管实时定量PCR检测方法，用于直接快速测定PMP22基因剂量。该方法涉及多重反应，使用来自PMP22外显子3的带有TAMRA淬灭剂的FAM标记的Taqman探针和来自白蛋白基因外显子12的带有非荧光淬灭剂的VIC标记的探针作为内部参照。PMP22基因的拷贝数通过比较阈值循环法（deltadeltaCt）确定。每个样本一式四份运行，并在两个不同的阈值水平进行分析。给出最小标准差的水平被记录下来。我们通过对252例已知基因型的HMSN患者进行回顾性分析来评估该方法，在99%的病例中证实了先前的发现。两名患者通过微卫星分析被误诊，而定量实时PCR确定了正确的基因型，FISH也证实了这一点。因此，该方法对微卫星分析显示出更高的灵敏度，并且具有对CMT1A和HNPP进行定量分析的快速且统一检测的额外优势。
