颅骨锁骨发育不全家系基因多态性与表型关系的研究

目的研究家族性颅锁骨发育不全家系基因型与临床表型的关系。方法提取临床收集的一个先天性颅锁骨发育不全家系中患者和健康成员外周血基因组DNA,PCR扩增RUNX2/CBFA1基因7个编码外显子及其侧翼内含子序列,分别进行正反向测序,对发现的突变位点行酶切分析验证。结果家系中两位患者（先证者及其父亲）显示cDNA 346T→A的杂合突变,使编码的色氨酸变成精氨酸（W 116R）,属错义突变。酶切结果进一步证实了该错义突变。测序还发现先证者父亲cDNA 198G→A的杂合突变,致第66位氨基酸的密码子GCG被GCA取代,但二者均编码丙氨酸,属同义突变。先证者及家系健康成员中未见此改变。结论 RUNX2/CBFA1基因346T→A杂合突变是该家系发病的分子基础。

Study of Genotype - phenetype Correlation for One Chinese Family with Cleidocranial Dysplasia

Objective This study aimed to explore the genotype-phenetype correlation for one chinese family with cleidocranial dysplasia（CCD）.Methods Genomic DNA was extracted from peripheral blood sample of each patient and healthy family member.The all 7 coding exons of RUNX2/CBFA1 and their flanking intronic sequences were amplified by polymerase chain reaction（PCR）,and direct bilateral sequencing of the PCR products was used to identify the possible mutations.The mutation was further confirmed by restriction analysis.Results Bilateral sequencing of PCR amplified fragments in proband and his father revealed a heterozygous T-to-A transition mutation at nucleotide 346 in exon 1 of RUNX2/CBFA1,which gave rise to amino acid substitutions of tryptophane（Trp,W） for arginine（Arg,R） at codon 116（W116R）.It was a missense mutation.Bilateral sequencing in proband’s father also revealed a novel G/A heterozygous polymorphism（G198A） at nucleotide 198 in exon 1,and the codon 66 was changed from GCG to GCA,both them encoding alanine（Ala,A）.It was synonymous mutation.This mutation did not appeared in the proband and healthy family members.The heterozygous T-to-A transition mutation at nucleotide 346 would removed the restriction site,so for the proband and his father in the first family,the nicking results of exon 1 would not only produce the two fragments,but also more of a non-cutting 513bp fragment,which was used to confirm the mutation.It was absent in healthy members.Conclusion The RUNX2/CBFA1 gene was responsible for the chinese families with CCD,and the 346T→A mutation was the molecular basis of the Chinese family with CCD.