Detection of mutations in the COL4A5 gene in over 90% of male patients with X-linked Alport's syndrome by RT-PCR and direct sequencing
Detection of mutations in the COL4A5 gene in over 90% of male patients with X-linked Alport's syndrome by RT-PCR and direct sequencing
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DOI：
10.1016/s0272-6386(99)70042-9
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发表时间：
1999-11-01
影响因子：
13.2
通讯作者：
Yoshikawa, N
中科院分区：
文献类型：
--
作者：
Inoue, YJ;Nishio, H;Yoshikawa, N
x连锁Alport综合征是由编码IV型胶原α 5链（α 5[IV]）的COL4A5基因突变引起的。基因组DMA上的聚合酶链反应-单str和构象多态性（PCR-SSCP）先前已被用于筛选COL4A5基因的突变，但这种方法相对不敏感，在不到50%的患者中检测到突变。在这里，我们报告了COL4A5基因的整个编码区域的系统分析，使用巢式逆转录聚合酶链反应（RT-PCR）和直接序列法使用白细胞。本研究检查了22例无亲缘关系的日本x连锁Alport综合征患者，这些患者在肾小球或表皮基底膜中表现出α 5（IV）的异常表达。13名男性患者中有12名（92%）和9名女性患者中有5名（56%）发现了预测为致病性的突变。6名患者有错义突变，4名有框外缺失突变，3名有无义突变，3名有导致转录本外显子丢失的突变。目前的研究表明，巢式RT-PCR和使用白细胞的直接序列法具有很高的敏感性，为x连锁Alport综合征患者的系统基因分析提供了一种有用的方法。(C) 1999年由国家肾脏基金会，Inc。
X-linked Alport's syndrome is caused by mutations in the COL4A5 gene encoding the type IV collagen alpha 5 chain (alpha 5[IV]). Polymerase chain reaction-single-str and conformation polymorphism (PCR-SSCP) on genomic DMA has previously been used to screen for mutations in the COL4A5 gene, but this method was relatively insensitive, with mutations detected in less than 50% of patients. Here, we report a systematic analysis of the entire coding region of the COL4A5 gene, using nested reverse-transcription-polymerase chain reaction (RT-PCR) and the direct sequence method using leukocytes. This study examines twenty-two unrelated Japanese patients with X-linked Alport's syndrome showing abnormal expression of alpha 5(IV) in the glomerular or epidermal basement membranes. Mutations that were predicted to be pathogenic were identified in 12 of the 13 male patients (92%) and five of the nine female patients (56%). Six patients had missense mutations, four had out-of-frame deletion mutations, three had nonsense mutations, and three had mutations causing exon loss of the, transcript. The current study shows that nested RT-PCR and the direct sequence method using leukocytes are highly sensitive and offer a useful approach for systematic gene analysis in patients with X-linked Alport's syndrome. (C) 1999 by the National Kidney Foundation, Inc.