[Analysis of allele frequencies of HLA-DRB1*12:01:01G and HLA-DRB1*14:01:01G groups].

OBJECTIVE: To discriminate and analyze the relative frequencies of alleles in HLA-DRB1*12:01:01G(HLA-DRB1*12:01:01/12:06/12:10/12:17) and HLA-DRB1*14:01:01G (DRB1*14:01:01/14:54) groups and assess their associations with HLA-DRB3 and HLA-DQB1 loci. METHODS: A total of 115 DNA samples previously typed as HLA-DRB1*12:01:01G and 108 samples from HLA-DRB1*14:01:01G were selected. DNA sequences for exons 1 to 3 of the HLA-DRB1 locus were analyzed for HLA-DRB1*12:01:01G, and exons 2 to 3 were analyzed for HLA-DRB1*14:01:01G by polymerase chain reaction sequence-based typing (PCR-SBT). Genotyping of HLA-DRB3 and HLA-DQB1 were achieved by PCR-SBT. RESULTS: Among 115 samples previously typed as HLA-DRB1*12:01:01G, 101 (87.8%) were confirmed as HLA-DRB1*12:01:01 and 14 (12.2%) were HLA-DRB1*12:10, but HLA-DRB1*12:06 and HLA-DRB1*12:17 alleles were not identified. For 108 samples previously typed as HLA-DRB1*14:01:01G, all were typed as HLA-DRB1*14:54. HLA-DRB1*12:01:01 was linked with HLA-DRB3*01:01:02 and HLA-DQB1*03:01, while HLA-DRB1*12:10 was strongly linked with HLA-DRB3*02:02:01 and HLA-DQB1*03:01. HLA-DRB1*14:54 was strongly linked with HLA-DRB3*02:02:01 and two different HLA-DQB1*05:02, *05:03 alleles. CONCLUSION: HLA-DRB1*12:01:01 was more prevalent than HLA-DRB1*12:10 in the HLA-DRB1*12:01:01G group, and HLA-DRB1*14:54 was the dominant allele for HLA-DRB1*14:01:01G.

[Recombination between human leukocyte antigen -A and -C loci within two Chinese Han families].

OBJECTIVE: To investigate the recombination events between human leukocyte antigen (HLA) loci within two families. METHODS: Identification of HLA-A, -C, -B, -DRB1 and -DQB1 loci was firstly carried out using polymerase chain reaction-sequence specific oligonucleotide. Then HLA high resolution typing was performed using polymerase chain reaction sequencing-based typing. The recombination between HLA loci was identified by family genetic analysis. The parentage possibility was analyzed by short tandom repeat technique. RESULTS: Recombination between the HLA-A and C loci was identified within two families. One individual inherited a paternal haplotype that was the result of a recombination event between the father's HLA-A and -C loci on his chromosomes. The other individual inherited a maternal haplotype that was the result of a recombination event between the mother's HLA-A and -C loci. The high parentage possibilities were obtained in the family members. CONCLUSION: The recombination events of HLA-A and -C have been found in two Chinese families, which may help further study on the mechanism of HLA recombination.

[Identification of a novel allele HLA-B*15:129 by polymerase chain reaction with allele group-specific primers].

OBJECTIVE: To analyze the sequence of the exons 2-4 of human leukocyte antigen (HLA) novel allele HLA-B*15:129. METHODS: DNA of the proband was extracted from whole blood by commercial DNA extraction kit. The amplification for HLA-B exons 2-4 was performed separately by polymerase chain reaction (PCR) with allele group specific primers. The PCR products were digested with enzymes and then directly sequenced for exons 2-4 of HLA-B locus in both directions. RESULTS: Sequencing results showed the HLA-B alleles of the proband included B*07:02 and a novel allele. The sequence of the novel allele has been submitted to GenBank (accession no. EF473219) and the allele has been officially named B*15:129 by the WHO Nomenclature Committee. Comparing with the HLA-B*15:01:01:01, the sequence of exons 2-4 of HLA-B*15:129 showed three nucleotide difference in exon 3 at positions 362 and 363 from GG to AT and positions 369 from C to T, which resulted in an amino acid change from Arg to Asn at codon 97. CONCLUSION: A novel HLA-B allele was identified and has been officially named B15:129 by the WHO Nomenclature Committee.

[Nucleotide sequence analysis of A novel HLA-B*15:124 allele confirmed].

This study was purposed to investigate the nucleotide sequences of a novel HLA-B*15:124 allele and its molecular mechanism. The genomic DNA from whole blood was extracted by using commercial DNA extraction kit. The sequences of exon 2, 3 and 4 of HLA-B locus in the proband were amplified by PCR with group-specific primers, the PCR products were purified by enzymes digestion, then exon 2 to 4 of HLA-B locus for both orientations was sequenced. The results showed that 2 HLA-B alleles of proband were gained after amplification and sequencing of group-specific primers, among them one was a B*40:03, another was a novel allele. After BLAST analysis, the novel allele showed nucleotides different from HLA-B*15:52 in exon 3 at nucleotide position 427 A > T and 440 G > T which resulted in amino acid change from Thr to Ser at codon 143 and Trp to Leu at conon 147. It is concluded that a novel HLA-B allele has two different nucleotides. This HLA-B allele is identified and has been officially named B*15:124 by the WHO Nomenclature Committee.

[Sequence analysis of a novel human leukocyte antigen allele HLA-A*9206].

OBJECTIVE: To investigate the molecular genetic basis for a human leukocyte antigen (HLA) novel allele HLA-A*9206 in the Chinese population. METHODS: DNA was extracted from whole blood by PEL-FREEZ DNA extraction kit. The amplification of HLA-A exons 1-8 of the proband was preformed and the PCR products were sequenced using ABI sequencing kit. Both strands of exons 2, 3 and 4 of the amplified product were sequenced. The polymerase chain reaction-sequence specific primer (PCR-SSP) was performed to split the two alleles apart and confirm the mutations detected by sequencing. RESULTS: The sequencing results showed that the HLA-A alleles of the proband were A*1101 and a novel allele. The sequence of the novel allele has been submitted to GenBank (EF062306). After Blast analysis, the novel allele shows one nucleotide different from the HLA-A*0206 in exon 3 at nucleotide position 530 (C to T). This results in an amino acid change from Ala to Val at codon 153. CONCLUSION: This allele is a novel allele and has been officially named A*9206 by the WHO Nomenclature Committee.

[Identification and sequence analysis of a novel HLA-B*3936 allele].

The aim of study was to confirm the novel HLA allele HLA-B*3936 in Chinese population and to analyze its sequence. The proband was a cord blood donor in the Zhejiang province. DNA was extracted from whole blood by PEL-FREEZ DNA extraction kit. The amplification of HLA-B exons 2 - 4 of the proband was performed by allele specific primer PCR and the amplified product was sequenced bidirectionally with primers. The sequencing results showed HLA-B alleles of the proband as B*4002 and the novel allele. The sequences of the novel allele have been submitted to GenBank (DQ242650, DQ242651, DQ242652). After Blast HLA analysis, the novel allele showed four nucleotide differences with HLA-B*3901 at nucleotide position 527 T-->A, 538 C-->T, 539 T-->G, 544 A-->G in exon 3. It resulted in three amino acid change from Val to Glu at codon 152, Ile to Trp at codon 156, Thr to Ala at codon 158. The result suggested that this allele is a novel allele and has been officially named HLA-B*3936 by the WHO Nomenclature Committee.

[Identification and sequence analysis of a null HLA-B allele HLA-B*5408N newly detected].

The study was purposed to investigate the molecular genetic basis for HLA novel allele HLA-B*5408N in Chinese population. DNA was extracted from whole blood by commercial DNA extraction kit, the HLA-B exons 2 - 4 of the proband was amplified by allele specific primers PCR and the amplified product was sequenced for exons 2, 3 and 4 bidirectionally. The sequencing results showed HLA-B alleles of the proband as B*1527 and the novel allele. The sequences of the novel allele have been submitted to Genbank (DQ295998, DQ295999, DQ296000). After blast analysis, the novel allele showed a single nucleotide mismatch with HLA-B*5401 in exon 3 at position 553 G-->T, which resulted in an amino acid changing from Glu to premature stop codon at position 161. No the HLA-B54 antigen specificity expression in the proband cells was found using HLA-AB serological Typing Trays. It is concluded that this allele is a novel null allele and has been officially named B*5408N by the WHO Nomenclature Committee.

[The genetic polymorphism of cytokine genes in Zhejiang Han individuals].

In order to investigate the genetic polymorphism of 13 cytokines genes in Han individuals from Zhejiang. The polymerase chain reaction-sequence specific primers (PCR-SSP) method was used to determine genetic polymorphisms in 100 unrelated healthy Han individuals from Zhejiang province. We found that: (1) The frequency of the TGFbeta (G codon 25) allele is 1.00 and no TGFbeta (C codon 25) allele is found. (2) The frequencies of IL-1alpha(C-889), IL-1beta(C +3962), IL-4 (T-1098), IL-6 (G-174), IL-6 (G nt565), IL-10 (A-1082) and TNFalpha (G-238) alleles are more than 0.95; while the frequencies of IL-1alpha (T-889), IL-1alpha (T +3962), IL-4 (G-1098), IL-6 (A-174), IL-6 (A nt565), IL-10 (G-1082), TNFalpha (A-238) alleles are less than 0.05. (3) The haplotype frequency of the high-producer-type GCC in IL-10 gene is 0.05, while the low-producer-type ATA is 0.735. The haplotype frequency of the high-producer-type TG in the TGFbeta gene is 0.49, while the low-producer-type CC is 0. The haplotype frequency of the high-producer-types AG and AA in the TNFalpha gene is 0.055, while of the low-producer-types GG and GA is 0.945. The results showed that the genetic polymorphism of cytokine genes in Han individuals from Zhejiang Province is distinctive.

[Sequence analysis of a novel HLA-A*3308 allele].

OBJECTIVE: To investigate the molecular genetics basis for HLA novel allele HLA-A*3308 in Chinese population. METHODS: DNA was extracted from whole blood by PEL-FREEZ DNA extraction kit. The amplification of HLA-A exons 1-8 of the proband was preformed by PCR and the amplified product was cloned with TOPO cloning sequencing kit to split the two alleles apart. Both strands of exons 2, 3 and 4 of chosen clones were sequenced. The PCR-SSP was performed to confirm the mutations detected by sequencing. RESULTS: The sequencing results showed HLA-A alleles of the proband as A*0201 and the novel allele. The sequences of the novel allele have been submitted to GenBank (DQ089631, DQ089632, DQ089633). BLAST analysis showed that the novel allele got the difference from A*3303 by five nucleotides at positions 240 A>T, 256C>G, 259A>G, 261C>G and 270T>A in exon 2. This resulted in three amino acids changes from Arg to Gly at codon 62, Asn to Glu at codon 63 and Asn to Lys at codon 66. CONCLUSION: This allele is a novel allele and has been officially named A*3308 by the WHO Nomenclature Committee.

[Sequence analysis of a novel HLA-DRB1 allele, DRB1 * 1212].

OBJECTIVE: To investigate the molecular genetics basis for HLA novel allele HLA-DRB1*1212 in Chinese population. METHODS: Genomic DNA was extracted from whole blood by salting-out method. HLA-DRB1 gene exon 2 was amplified by PCR with group-specific primers from genomic DNA. PCR products were cut back from agarose gels and purified to sequence directly. The polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSO) was performed to confirm the mutations which were detected by sequencing in this study. RESULTS: The sequencing results showed HLA-DRB1 alleles of the proband as DRB1*090102 and the novel allele. The sequences of the novel allele have been submitted to GenBank (AY899825). Through BLAST analysis, the novel allele was found to be different from DRB1*120101 at position 199A-->C in exon 2, that results in an amino acid change from Ile to Leu at codon 67. CONCLUSION: This allele is a novel and has been officially named as DRB1*1212 by the WHO Nomenclature Committee.

[Sequence analysis of HLA-B*4061 allele newly found].

The aim of this study was aimed to investigate the molecular genetic basis for a novel HLA allele, HLA-B*4061, in Chinese population. DNA was extracted from whole blood by salting-out method. The HLA-B exons 1 - 8 of the proband was amplified and the amplified product was cloned using TOPO TA cloning sequencing kit to split the two alleles apart. Both strands of exons 2, 3 and 4 of chosen colonies were sequencing. The PCR-SSP was performed to confirm the mutations detected by sequencing. The sequencing results showed HLA-B alleles of the proband as B*4601 and the novel allele. The sequences of the novel allele have been submitted to GenBank (DQ089628, DQ089629, DQ089630). After HLA blast analysis, the novel allele showed a single nucleotide mismatch with B*400101 in exon 2 at position 272 C-->A, as the results, changing amino acid from Ser to Tyr at codon 67. It is concluded that this allele is a novel one and has been officially named B*4061 by the WHO Nomenclature Committee.