[An analysis of the reason for HLA-C allele dropout in five samples by sequence-based typing].
OBJECTIVE
To analyze the possible reason for HLA-C allele dropout in routine sequence-based typing (SBT) and improve the accuracy of HLA-C SBT test.


METHODS
A total of 620 randomly selected samples from healthy voluntary blood donors in Shenzhen were typed at HLA-C locus by sequence-based typing using the AlleleSEQR HLA-C plus sequence-based typing kit. Samples with no full match result were subjected to cloning and haplotype sequencing of the full-length HLA-C gene. If no novel mutations were found, samples were then retyped, using our self-designed PCR primer pair and PCR conditions replacing the AlleleSEQR HLA-C PCR reagents in the PCR set-up procedure so as to analyze the potential reasons for causing abnormal SBT result.


RESULTS
In the 620 samples typed at HLA-C locus using the AlleleSEQR HLA-C SBT commercial kit, 5 samples with no full match result were identified. The closest genotype showed one nucleotide mismatch with many different allele groups at different nucleotide position. Based on the PCR-SBT nucleotide sequence, heterozygous nucleotides were determined only in exon 4, whereas the nucleotides in exon 2 and 3 were all homozygotes. The results showed that HLA-Cw*0706 allele dropout existed in all the 5 samples with abnormal SBT results initially identified by AlleleSEQR HLA-C SBT kit, no novel mutation was found.


CONCLUSIONS
The results indicate that the PCR primer pair incompatible with DNA template may result in allele dropout in HLA-C SBT test. Based on the characterization of HLA-C full-length, it is essential to develop HLA-C SBT kit suitable for Chinese population in the future.
Zeng
Jian-qiang
JQ
Shenzhen Blood Center, Shenzhen Key Laboratory of Histocompatibility and Immunogenetics, Shenzhen, Guangdong, 518035, PR China.
Xu
Yun-ping
YP
Wang
Da-ming
DM
Zou
Hong-yan
HY
Deng
Zhi-hui
ZH
Yang
Bao-cheng
BC
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods / Q000592:standards; D015236:HLA-C Antigens / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards*
D015236:HLA-C Antigens