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目的对单纯腭裂并全面性癫伴热性惊厥附加症家系进行基因定位,并对候选基因GABRD进行测序研究。方法对家系进行全基因组扫描两点间连锁分析,设计GABRD基因9对外显子-内含子交界处内含子引物,PCR扩增患者及健康对照GABRD基因组序列,测序采用Sanger双脱氧链终止法,PCR产物直接测序,对比分析患者的突变序列及健康对照序列。结果两点连锁分析在1p36区域最大LOD值为1.68。GABRD基因测序显示第4外显子患者的序列分别为T/C和T/T,健康对照的序列分别为T/T和A/C。编码序列分析GGT、GGC及GGA均为甘氨酸。患者GABRD基因mRNA碱基序列为C425T多态性。GABRD第7外显子患者的序列分别为C/T和T/T,健康对照分别为T/T和C/C;异常序列位于mRNA第911碱基处,编码分析AGT及AGC均编码丝氨酸。患者的碱基序列出现了T911C多态性。结论单纯腭裂并全面性癫伴热性惊厥附加症致病基因在1p36区域取得一定的连锁关系,在该家系未发现突变基因;所发现的C425T和T911C单个碱基多态性为其他腭裂及癫家系的连锁分析、关联分析及分子遗传学研究提供了依据。
Objective To investigate the gene localization of pedigrees with simple palate and generalized epilepsy with febrile seizures, and to sequence the candidate gene GABRD. Methods A genome-wide linkage scan was performed between the two families. The GABRD gene 9 intron-intron junction intronic primers were designed. The GABRD gene sequence was amplified by PCR and sequenced by Sanger dideoxy chain termination PCR products were sequenced directly, and the patient’s mutation sequence and healthy control sequence were compared. Results The maximum LOD value of the two-point linkage analysis in the 1p36 region was 1.68. GABRD gene sequencing showed that the sequence of the fourth exon was T / C and T / T, respectively. The sequences of healthy controls were T / T and A / C respectively. Coding sequence analysis GGT, GGC and GGA are glycine. Patients GABRD gene mRNA base sequence C425T polymorphism. The sequences of GABRD exon 7 were C / T and T / T respectively, and the healthy controls were T / T and C / C respectively. The abnormal sequence was located at the 911th base of mRNA. The encoding AGT and AGC encoded serine. The patient’s nucleotide sequence showed a T911C polymorphism. Conclusions There is a certain linkage relationship between pathogenic genes in 1p36 region of pure cleft palate and generalized epilepsy with febrile seizures, and no mutant gene was found in this pedigree. The single base polymorphisms of C425T and T911C found were other cleft palate and Epilepsy pedigree linkage analysis, association analysis and molecular genetics provide the basis for the study.