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目的:通过亚铁螯合酶(FECH)基因突变检测和核苷酸多态性分析确诊疾病,分析中国人群中红细胞生成性原卟啉病(EPP)的基因谱。创新点:通过FECH基因检测和多态性分析可精准诊断EPP这一罕见病,可减少漏诊误诊。另外,本研究扩充了中国EPP患者FECH基因突变谱。方法:选取北京协和医院就诊疑似EPP患者两例,采用聚合酶链反应(PCR)扩增FECH基因和5-氨基酮戊酸合成酶(ALAS2)基因并进行测序,同时在美国国立生物技术信息中心(NCBI)网站中比对基因突变情况,并行单核苷酸多态性(SNP)分析;通过荧光分光光度计检测其血浆中荧光激发峰值;检测血细胞内游离原卟啉浓度;临床评估皮肤对日光的光敏性。并对1名患者的FECH基因突变和SNP进行家系分析。结论:患者A的FECH基因cD NA中第973位碱基A缺失,造成病人的氨基酸序列从324位后发生框移突变;患者B的FECH基因cD NA中第1232位G>T突变,造成病人411位的半胱氨酸转变为苯丙氨酸(图3)。结合两例患者皮肤光敏性损害(图1)、游离原卟啉增高、血浆荧光激发峰值在630~634 nm处出现(图2)和ALAS2基因未突变的特征,明确诊断为EPP。同时发现多个核苷酸突变,包括c.798 C>G、c.921 A>G、IVS1-23 C>T、IVS3+23 A>G、IVS9+35 C>T和IVS3-48 T>C(表1)。本研究通过基因鉴定和SNP分析,结合临床特征可精准诊断EPP这一罕见病,并扩充了中国EPP患者FECH基因突变谱。
OBJECTIVE: To determine the genetic susceptibility of erythropoietic protoporphyrins (EPP) in Chinese population through the detection of mutations in the ferrochelatase (FECH) gene and analysis of nucleotide polymorphisms. Innovative point: Through the FECH gene detection and polymorphism analysis can accurately diagnose the rare case of EPP can reduce misdiagnosis. In addition, we expanded the FECH gene mutation profile in Chinese EPP patients. Methods: Two patients with suspected EPP in Peking Union Medical College Hospital were enrolled in this study. The FECH gene and 5-aminolevulinic acid synthase (ALAS2) gene were amplified by polymerase chain reaction (PCR) and sequenced. At the same time, the National Center for Biotechnology Information (NCBI), and analyzed the SNP in parallel. Fluorescence peak of plasma was detected by fluorescence spectrophotometer. The concentration of free protoporphyrin in blood was detected. Light sensitivity of daylight. A family analysis of FECH gene mutations and SNPs in one patient was performed. CONCLUSION: The deletion of base A at position 973 of cD NA of FECH gene of patient A resulted in the frame shift mutation of amino acid sequence of patient from position 324; the mutation of position 1232 G> T in cD NA of FECH gene of patient B resulted in the patient The cysteine at position 411 was converted to phenylalanine (Figure 3). Combined with the photosensitivity of the skin of two patients (Fig. 1), free protoporphyrin increased, peak plasma fluorescence appeared at 630-634 nm (Figure 2), and the non-mutated ALAS2 gene was characterized as a clear diagnosis of EPP. Multiple nucleotide mutations were found at the same time, including c.798 C> G, c.921 A> G, IVS1-23 C> T, IVS3 + 23 A> G, IVS9 + 35 C> T and IVS3-48 T> C (Table 1). In this study, we can accurately diagnose the rare case of EPP through gene identification and SNP analysis combined with clinical features and expand the FECH gene mutation profile in Chinese EPP patients.