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目的:通过对Gordon综合征(PHA2)患者的WNK4和WNK1基因外显子扩增测序,研究我国PHA2患者WNK基因的突变特点。方法:通过查阅住院病史,收集不明原因高血压伴高血钾而肾功能正常的患者,进行临床和实验室检查,确诊PHA2。采集患病者及患病/非患病亲属的外周血,抽提DNA,设计17对引物扩增WNK4的19个外显子,27对引物扩增WNK1的28个外显子,纯化后送测序,测序结果进行序列比对。结果:发现PHA2患者4例,其中2例表现为家族遗传性,另2例为散发。先证者及所有患病家系成员共8人,年龄6～79岁,均表现为不明原因的高血压、高血钾,肾功能正常。8例中的5例伴有肾小管酸中毒,6例伴有高氯血症。DNA扩增后测序比对发现,其中1例散发患者为WNK4基因的杂合错义突变,位于17号外显子,造成氨基酸序列的改变,赖氨酸突变为谷氨酸(K1169E)。对其2个非患病女儿以及200个正常对照的WNK4基因检测均未发现该突变。其它2个患病家系及1个散发患者未发现WNK4基因突变,所有患者和健康者均未发现WNK1外显子突变。结论:PHA2是一种少见的单基因遗传疾病,漏误诊率很高。已报道的WNK4致病位点均位于螺旋-螺旋结构域附近。我们发现的K1169E为HGMD未收录的新突变,也位于螺旋-螺旋结构域,该位点氨基酸在不同物种高度保守,而且该位点氨基酸的变化伴有电荷的改变,可能导致蛋白质构象的改变。
OBJECTIVE: To study the mutation of WNK gene in Chinese PHA2 patients by exon sequencing of WNK4 and WNK1 gene in patients with PHA2. Methods: Clinical and laboratory tests were performed to determine PHA2 by referring to the history of hospitalization, collecting patients with unexplained hypertension associated with hyperkalemia and normal renal function. A total of 17 pairs of primers were designed to amplify 19 exons of WNK4 and 27 pairs of primers to amplify 28 exons of WNK1. Sequencing, sequencing results for sequence alignment. Results: Four patients with PHA2 were found, of which two showed familial heredity and the other two were exuded. The proband and all affected family members a total of 8 people, aged 6 to 79 years old, showed unexplained hypertension, hyperkalemia, normal renal function. Five of the eight patients had tubular acidosis and six had hyperchloremia. After DNA amplification, it was found that one case of sporadic patients was heterozygous missense mutation of WNK4 gene located on exon 17, resulting in the change of amino acid sequence, and the mutation of lysine to glutamic acid (K1169E). No mutation was detected in WNK4 gene of two non-diseased daughters and 200 normal controls. No other WNK4 gene mutation was found in the other two affected families and one sporadic patient. No mutation of WNK1 was detected in all patients and healthy controls. Conclusion: PHA2 is a rare single-gene genetic disease, misdiagnosis rate is high. Reported WNK4 pathogenic sites are located near the helix-helix domain. We found that K1169E is a new mutation that is not included in HGMD. It is also located in the helix-helix domain. The amino acids in this site are highly conserved in different species. Moreover, changes in amino acids at this site with changes in charge may lead to changes in protein conformation.