【作者】 张敏；

【导师】 兰风华；

【作者基本信息】 福建医科大学 ， 临床检验诊断学， 2014， 硕士

【摘要】 家族性腺瘤性息肉病（familial adenomatous polyposis, FAP）是以结直肠内生长大量腺瘤性息肉为主要特征的一种常染色体显性遗传病。根据大肠息肉数和发病年龄可分为息肉数多于100枚，具有较早发病年龄的经典型家族性腺瘤性息肉病（classical familial adenomatous polyposis, CFAP）和息肉数少于100枚，具有较晚发病年龄的轻表型家族性腺瘤性息肉病（attenuated familialadenomatous polyposis, AFAP）。FAP主要由腺瘤性息肉病基因（adenomatouspolyposis coli, APC）突变所致，该基因定位于染色体5q21-22，长108kb，共有15个外显子，其中15号外显子占编码序列的77%，基因共编码含2843个氨基酸残基的蛋白质。APC蛋白具有和多种蛋白结合的位点，在负性调控Wnt/β-连环蛋白通路从而抑制结直肠肿瘤形成中起重要作用，APC蛋白通过下调β-连环蛋白，阻止其过多进入细胞核，若APC功能丧失，β-连环蛋白将在细胞质内堆积，并过多转移至细胞核促进癌基因表达。80-90%CFAP患者携带APC基因突变，其中大部分为无义突变和移码突变，其余还包括大片段缺失/重复，剪接位点处突变等，这些突变导致功能有缺陷的蛋白形成，从而引起疾病发生。本研究从DNA途径，对6个通过临床资料分析符合CFAP临床诊断的家系进行分子诊断，分34个片段扩增APC基因的15个外显子并对其PCR产物进行直接测序，结果显示6个CFAP家系患者均存在APC基因突变，其中家系1在APC基因15号外显子发现杂合性突变：c.2891T＞G（L964X），此突变导致密码子964处由亮氨酸密码子变为终止密码子；家系2在APC基因15号外显子发现杂合性突变：c.4012C＞T（Q1338X），此突变造成密码子1338处由谷氨酰胺密码子变为终止密码子；家系3在APC基因8号外显子发现杂合性突变：c.904C＞T（R302X），此突变造成密码子302处由精氨酸密码子变为终止密码子；家系4在APC基因15号外显子发现杂合性缺失突变：c.3927₃931delAAAGA，此突变造成碱基从密码子1309处出现移码，在密码子1312处形成终止密码子；家系5在APC基因15号外显子发现杂合性缺失突变：c.2154₂157delGAAA，此突变造成碱基从密码子690处出现移码，在密码子716处形成终止密码子；家系6在APC基因6号外显子发现杂合性突变：c.646C＞T（R216X），此突变造成密码子216处由精氨酸密码子变为终止密码子。上述6个家系APC基因突变均导致终止密码子提前出现，从而形成功能障碍的截短蛋白。通过人类基因突变数据库（The Human Gene Mutation Database, HGMD）查询，家系1：c.2891T＞G（L964X）和家系5：c.2154₂157delGAAA突变尚未见报道，为新的突变，其余在HGMD中均有记录，为已知突变。通过分子诊断，对FAP患者及其家庭成员进行APC基因检测，明确基因突变位点和类型，可为遗传咨询和产前诊断提供诊断依据，也可辅助临床科室更早、更准确地对患者进行诊断和治疗。更多还原

【Abstract】 Familial adenomatous polyposis （FAP） characterized by the numerousadenomatous polyps in the colon and rectum, is inherited in an autosomal dominantmanner. According to polyp number and age at onset, the phenotype is usuallyclassified as classical familial adenomatous polyposis （CFAP） and attenuated familialadenomatous polyposis （AFAP）. CFAP is characterized by more than100colorectaladenomas and early onset, and AFAP by fewer than100adenomas and a later age ofonset. FAP is mainly caused by the mutation in adenomatous polyposis coli （APC）gene. The APC gene spans108kb of genomic DNA, and is located at chromosome5q21-22. It contains15coding exons and encodes a protein containing2843aminoacids. The APC protein has multiple domains, through which it binds to variousproteins. It functions as a negative regulator of β-catenin levels and suppressescanonical Wnt signalling, which is essential for tumorigenesis. In the absence of theAPC protein, β-catenin accumulates in the nucleus and interacts with factors thatup-regulate the transcription of genes involved in cell cycle entry and progression,and accelerates the oncogene expression. APC mutations are found in approximately80%to90%of patients with CFAP, most of pathogenic alterations are nonsense orframeshift mutations, and large deletions and/or duplications, splice site mutations arealso pathogenic factors of FAP, which result in the formation of premature terminationof the protein of disordered function, and causes the disease.Gene mutation detection in six pedigrees with CFAP were studied at genomic DNA leavel in this study. Thirty-four fragments covering the entire coding sequenceof the APC gene were amplified by PCR, and the PCR products were detected bySanger sequencing. Gene mutations were found in all six pedigrees. The mutation ofc.2891T＞G（L964X） in APC exon15was detected in pedigree one, which changedcodon for leucine to termination codon at codon964. The mutation of c.4012C＞T（Q1338X）of exon15was detected in pedigree two, which altered the codon forGlutamine to termination codon at codon1338. The mutation of c.904C＞T（R302X）of exon8was detected in pedigree three, which resulted arginine codon to terminationcodon at codon302. The mutation of c.3927₃931delAAAGA of exon15wasdetected in pedigree four, leading to premature termination at codon1312. Themutation of c.2154₂157delGAAA of exon15was detected in pedigree five, leadingto premature termination at codon716. The mutation of c.646C＞T（R216X）of exon6was detected in pedigree six, changing the codon for arginine to termination codonat codon216. All mutations were searched for in the Human Gene Mutation Database（HGMD）, and mutations in pedigrees one and five were novel.Molecular diagnosis can reveal the mutions of APC gene and the genotypes ofthe family members. It can also provide reliable data for genetic consultation andprenatal diagnosis, and contributes clinically earlier diagnosis and more accuratetreatment to the affected individuals.更多还原