Wine J J, Kuo E, Hurlock G, Moss R B
Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California, USA.
Pediatrics. 2001 Feb;107(2):280-6. doi: 10.1542/peds.107.2.280.
The identities of a cystic fibrosis (CF) patient's CFTR mutations can influence therapeutic strategies, but because >800 CFTR mutations exist, cost-effective, comprehensive screening requires a multistage approach. Single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA) can be an important part of mutation detection, but must be calibrated within each laboratory. The sensitivity of a combined commercial-SSCP/HA approach to genotyping in a large, ethnically diverse US center CF population has not been established.
We screened all 27 CFTR exons in 10 human participants who had an unequivocal CF diagnosis including a positive sweat chloride test and at least 1 unknown allele after commercial testing for the 70 most common mutations by SSCP/HA. These participants were compared with 7 participants who had negative sweat tests but at least 1 other CF-like symptom meriting complete genotyping.
For the 10 CF participants, we detected 11 of 16 unknown alleles (69%) and all 4 of the known alleles (100%), for an overall rate of 75% inpatients not fully genotyped by conventional 70 mutation screen. For 7 participants with negative sweat tests, we confirmed 1 identified mutation in 14 alleles and detected 3 additional mutations. Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, DeltaF508, 451-458Delta8 bp, 5T, 663DeltaT, exon 13 frameshift, 1261+1G-->A and 3272-26A-->G). Three of these mutations were novel (G970D, L1093P, and 451-458Delta8 bp(1)). Thirteen other changes were detected, including the novel changes 1812-3 ins T, 4096-278 ins T, 4096-265 ins TG, and 4096-180 T-->G.
When combined with the 70 mutation Genzyme test, SSCP/HA analysis allows for detection of >95% of the mutations in an ethnically heterogeneous CF center population. We discuss 5 possible explanations that could account for the few remaining undetected mutations.
囊性纤维化（CF）患者CFTR突变的类型会影响治疗策略，但由于存在800多种CFTR突变，具有成本效益的全面筛查需要采用多阶段方法。单链构象多态性和异源双链分析（SSCP/HA）可以是突变检测的重要组成部分，但必须在每个实验室进行校准。在美国一个种族多样化的大型CF中心人群中，联合商业SSCP/HA方法进行基因分型的敏感性尚未确定。
我们对10名明确诊断为CF的人类参与者的所有27个CFTR外显子进行了筛查，这些参与者的汗液氯化物试验呈阳性，并且在通过SSCP/HA对70种最常见突变进行商业检测后至少有1个未知等位基因。将这些参与者与7名汗液试验阴性但至少有1种其他CF样症状且值得进行完整基因分型的参与者进行比较。
对于10名CF参与者，我们检测到了16个未知等位基因中的11个（69%）和所有4个已知等位基因（100%），对于未通过传统70突变筛查进行完全基因分型的住院患者，总体检出率为75%。对于7名汗液试验阴性的参与者，我们在14个等位基因中确认了1个已识别的突变，并检测到另外3个突变。两组中检测到的突变包括7个错义突变（S13F、P67L、G98R、S492F、G970D、L1093P、N1303K）和9个缺失、移码、无义或剪接突变（R75X、G542X、DeltaF508、451 - 458Delta8 bp、5T、663DeltaT、外显子13移码、1261 + 1G→A和3272 - 26A→G）。这些突变中有3个是新的（G970D、L1093P和451 - 458Delta8 bp(1)）。还检测到了其他13种变化，包括新变化1812 - 3 ins T、4096 - 278 ins T、4096 - 265 ins TG和4096 - 180 T→G。
当与70突变的健赞检测相结合时，SSCP/HA分析能够检测出种族异质的CF中心人群中>95%的突变。我们讨论了5种可能解释剩余少数未检测到的突变的原因。
