Detection of LDL receptor gene mutation in a child patient with familial hypercholesterolemia by whole cDNA sequence analysis

Objective To establish the whole cDNA sequencing method for analyzing the mutation of low-density lipoprotein receptor(LDLR) gene in a child patient with familial hypercholesterolemia(FH). Methods Seven pairs of primers for LDLR gene were designed and verified. One child patient with FH was performed medical examination,and the family was surveyed. The patient's peripheral blood sample was collected,and DNA and RNA were isolated. DNA was directly amplified by PCR with primers for LDLR gene and the amplified product was sequenced to find mutation sites. RNA was first reversed to be cDNA,then amplified by PCR with primers for LDLR gene,and the amplified product was performed sequencing analysis. The mutation sites were identified by comparing with normal LDLR gene sequence in GenBank. Finally the results obtained by the whole cDNA sequencing method were compared with the results by traditional DNA sequencing. Results Analysis for whole LDLR cDNA sequencing showed that LDLR gene contained 2 583base pairs,which was in concordance with the standard sequence of LDLR gene. The child patient was diagnosed as FH homozygote.The results obtained by the whole cDNA sequencing method were consistent with the results by traditional DNA sequencing,that is,both of them contained the terminator codon mutation in exon 2 and the single base substitution and the frame shift mutations in exon 6.Conclusion The whole cDNA sequencing method could be used to detect the mutations of LDLR gene in FH patients,which may provide the evidence for the gene diagnosis of FH.