Warshawsky Ilka, Chernova Olga B, Hübner Christian A, Stindl Reinhard, Henneke Marco, Gal Andreas, Natowicz Marvin R
Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Clin Chem. 2006 Jul;52(7):1267-75. doi: 10.1373/clinchem.2006.067967. Epub 2006 Apr 27.
Pelizaeus-Merzbacher disease is a rare X-linked neurodegenerative disorder caused by sequence variations in the proteolipid protein 1 gene (PLP1). PLP1 gene duplications account for approximately 50%-75% of cases and point variations for approximately 15%-20% of cases; deletions and insertions occur infrequently. We used multiplex ligation-dependent probe amplification (MLPA) to detect PLP1 gene alterations, especially gene duplications and deletions.
We performed MLPA on 102 samples from individuals with diverse PLP1 gene abnormalities and from controls, including 50 samples previously characterized for the PLP1 gene by quantitative PCR but which were anonymized for prior results and sex.
All males with PLP1 gene duplications (n = 13), 1 male with a triplication, 2 males with whole gene deletions, and all controls (n = 72) were unambiguously assigned to their correct genotype. Of 4 female carriers tested by MLPA and quantitative PCR, 3 were duplication carriers by both methods, and 1 was a triplication carrier by MLPA and a duplication carrier by quantitative PCR. For 1 sample with a partial deletion, MLPA showed exon 3 deleted but PCR showed exons 3 and 4 deleted. Sequence analysis of 2 samples with reduced dosage for exons 3 and 5 revealed point variations overlapping the annealing site for the corresponding MLPA probe. The precision of MLPA analysis was excellent and comparable to or better than quantitative PCR, with CVs of 4.3%-9.8%.
MLPA is a rapid and reliable method to determine PLP1 gene copies. Samples with partial PLP1 gene dosage alterations require confirmation with a non-MLPA method.
佩利措伊斯-梅茨巴赫病是一种罕见的X连锁神经退行性疾病，由蛋白脂质蛋白1基因（PLP1）的序列变异引起。PLP1基因重复约占病例的50%-75%，点变异约占病例的15%-20%；缺失和插入很少发生。我们使用多重连接依赖探针扩增（MLPA）来检测PLP1基因改变，尤其是基因重复和缺失。
我们对102个样本进行了MLPA检测，这些样本来自具有不同PLP1基因异常的个体和对照组，其中50个样本先前通过定量PCR对PLP1基因进行了特征分析，但为了保护先前结果和性别信息而进行了匿名处理。
所有携带PLP1基因重复的男性（n = 13）、1名携带三倍体的男性、2名携带全基因缺失的男性以及所有对照组（n = 72）都被明确无误地归为其正确的基因型。在通过MLPA和定量PCR检测的4名女性携带者中，3名通过两种方法均为重复携带者，1名通过MLPA为三倍体携带者，通过定量PCR为重复携带者。对于1个部分缺失的样本，MLPA显示外显子3缺失，但PCR显示外显子3和4缺失。对2个外显子3和5剂量减少的样本进行序列分析，发现点变异与相应MLPA探针的退火位点重叠。MLPA分析的精密度极佳，与定量PCR相当或更好，变异系数为4.3%-9.8%。
MLPA是一种快速且可靠的确定PLP1基因拷贝数的方法。PLP1基因剂量部分改变的样本需要用非MLPA方法进行确认。
