Sequence-based Typing Assay Research Articles

Establishment of sequence-based typing assay for KIR2DS4 gene and identification of a new allele KIR2DS4*016

To establish a reliable sequence-based typing method for KIR2DS4 and study its allele polymorphism in Chinese Han population. Using PCR-SSP method to detect the positive or negative of KIR2DS4 gene in 222 random Chinese Han individuals, and then using the method of high fidelity and long-fragment PCR-SBT to amplify, sequence and genotype the exons 4 and 5 of KIR2DS4 positive individuals. We successfully amplified the fragment with 3.2 kb length contains exons 4 and 5 of KIR2DS4 and detected the KIR2DS4 allele frequency in Chinese Han population. 209 KIR2DS4 positive individuals were detected, and the positive rate is 94.1%. By sequence-based typing, we identified 12 genotypes and 7 alleles of KIR2DS4. The 6 known alleles and their detection frequency is as follows: KIR2DS4* 00101/011 (180, 81.1%), KIR2DS4* 010 (53, 23.9%), KIR2DS4* 004 (34, 15.3%), KIR2DS4* 003 (15 and 6.8%), KIR2DS4* 006 (2, 0.9%) and KIR2DS4* 015 (1, 0.5%). In this study, we found a new allele, KIR2DS4* 016, with the difference in exon 5 comparing its most similar allele KIR2DS4* 010. In the exon 5 of KIR2DS4* 010, there is a 22bp-deletion, while the exon 5 of KIR2DS4* 016 is normal. This is not a rare allele because it was detected 3 times in studied population and with the frequency of 1.4%. The sequence of the new allele sequence has been submitted to GenBank (accession no.: KC414890) and the IPD -KIR database (submission no.: IWS40001804), and was nominated by WHO nomenclature committee for HLA system. In this study, a sequence-based typing method for KIR2DS4 was established, and the polymorphism data of KIR2DS4 in Chinese Han population was enriched by studying the allele polymorphism and new allele.

Short communication: Identification of variation in the ovine prolactin gene of Chios sheep with a cost-effective sequence-based typing assay

The present study identified single nucleotide polymorphisms (SNP) in the coding and untranslated regions of the ovine prolactin gene of Chios sheep. By developing a cost-effective direct sequence-based typing assay, around 600 bp of reliable sequencing data and clear identification of heterozygous positions was achieved. Five SNP were found, located in exons 2 (KC764410:g.567G>A, g.625C>T, g.683C>A) and 3 (KC764410:g.2015C>A, g.2101G>A), whereas the remaining exons were monomorphic. The identified SNP were synonymous, with the exception of the g.567G>A SNP, which results in an Arg to His amino acid change. As the sequencing cost of the sequence-based typing assay was 20 orders of magnitude lower compared with a standard Sanger method, the assay was also used as a genotyping tool. The identified polymorphism was genotyped for 247 ewes and was subsequently used in mixed model association analyses of milk yield, milk fat content, and litter size at birth. The association analysis revealed a significant dominance effect of 0.17 ± 0.07 of the g.2015C>A SNP on milk fat percentage, whereas a dominance effect of -21.33 ± 10.51 of the same SNP on total lactation milk yield was also estimated. The g.2015C>A SNP explained 2.47 and 3.68% of the total phenotypic variance of milk yield and milk fat percentage, respectively, whereas the corresponding values for the animal variance were 7.14 and 11.75%. A suggestive association of the nonsynonymous g.567G>A SNP with litter size at birth was also detected.

Resolution of ambiguous HLA genotyping in korean by multi-group-specific sequence-based typing.

PurposeTo evaluate a multi-group-specific sequence-based typing (SBT) method for resolving ambiguous results from human leukocyte antigen (HLA) genotyping.Materials and MethodsA total of 50 samples that showed ambiguous genotypes for at least two HLA loci from HLA-A, -B, -C and -DRB1 by the conventional SBT assay were evaluated using a new SBT test, the AVITA plus assay. The most likely HLA genotypes for the respective samples considering allele frequencies in Korean were concordant between the AVITA and conventional SBT assays.ResultsAn average of 3.3 loci among the HLA-A, -B, -C and -DRB1 loci per sample gave results with two or more possible allele combinations with the conventional SBT, and 48 (96.0%) out of 50 showed reduced numbers of possible genotypes for at least one HLA locus with the AVITA. A total of 41, 43, 42, and 38 cases among the 50 samples showed ambiguous results for HLA-A, -B, -C, and -DRB1 typing by the conventional SBT, respectively. The average numbers of possible allele combinations for the respective four HLA loci were 8.2, 6.7, 5.9, and 3.2, and they were reduced to 1.5, 2.2, 4.4, and 1.8, respectively, by the AVITA. Ambiguity was resolved by the AVITA in 33 (80.5%), 31 (72.1%), 17 (40.5%) and 28 (73.7%) samples among the ambiguous cases from the conventional SBT for HLA-A, -B, -C, and -DRB1 typing, respectively.ConclusionThe multi-group-specific SBT method considerably reduced the number of ambiguous results, and thus may be useful for accurate HLA typing in clinical laboratories.

Human leukocyte antigen class I supertypes and viral control in HIV-1 infected former plasma donors from China

Background The role of human leukocyte antigen (HLA) class I supertypes in controlling human immunodeficiency virus type 1 (HIV-1) infection in Chinese has not been established. The aim of this study is to examine the frequency of HLA-A and HLA-B alleles and supertypes of 222 HIV-1 infected former plasma donors in central China and to investigate their impact on HIV-1 viral control. Methods HLA-A and HLA-B alleles were genotyped with PCR-SSP and sequence-based typing assay to four-digit resolution. The HLA alleles were classified functionally to 4 HLA-A supertypes and 6 HLA-B supertypes according to their shared peptide binding properties. Plasma viral load was determined using the Roche Amplicor ultrasensitive assay which has a lower detection limit of 50 copies HIV-1 RNA per ml. Results HLA-A03 supertypes(A03s) and HLA-B62 supertypes (B62s) were associated with lower viral load (P=0.0206, P=0.0483), whereas HLA- A24 supertypes(A24s) appeared to have an association with higher viral load (P=0.0483). There was ah ighly significant correlation between the genotypic supertypes(GS) and viral load (Kendall’s tau b = 0.180, P=0.000). The median viral load was lower among A*3001(P=0.0139), A*1101(P=0.0096), B*5101(P=0.0025), B*3501(P=0.0091) or B*4601(P=0.001) carriers and higher in A*2301(P=0.0106) carriers. Conclusion

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The FDA has issued new draft guidance on molecular testing around Research Use Only (RUO) and non-Quality System Regulation (QSR) products in clinical labs. Promega has introduced a new QSR-compliant nucleic acid extractor and kit specifically to address this issue. At BloodCenter of Wisconsin we have evaluated this new extractor, the Maxwell ® CSC, in terms of its performance in HLA typing assays as well as its fit in the molecular HLA workflow. Patient samples (buccal swabs) were extracted using the Maxwell ® CSC, and then evaluated for total yield and purity of DNA. They were compared to the existing extraction method and evaluated by sequence-based typing, rSSO and STR assays for overall success and results concordance. The data handling capabilities of the instrument were also evaluated, such as how the run reports affected technician time, downstream operations and integration with electronic records. DNA concentrations from double-buccal swab isolations ranged from 13 ng/μl to 247 ng/μl, with a mean concentration of 76.31 ng/μl. DNA purities ranged from OD260/280 ratios of 1.69 to 2.00, with a mean of 1.89. These results are comparable to those obtained on the Maxwell ® MDx instrument in our laboratory. All samples tested in downstream HLA STR assay, KIR rSSO, HLA rSSO and sequence-based typing assays were successful and concordant with previously obtained results. The Maxwell ® CSC is a QSR-compliant system which will allow molecular laboratories to adhere to the draft FDA guidance for molecular testing. It provides a solution for nucleic acid extraction which performs equivalently to Promega’s current extraction systems with regards to DNA yield, quality and utility in downstream assays. The Maxwell® CSC also helps reduce data entry tasks with exportable sample-specific results. This is useful for tracking samples throughout the workflow, reducing the need to re-enter sample identifiers, saving technicians time and reducing errors.

The HLA‐DRB1 gene and Graves disease in Taiwanese children: a case‐control and family‐based study

Graves disease (GD) is an autoimmune thyroid disease with a female preponderance and a wide range of ages at onset, and human leukocyte antigen (HLA) gene plays a primary role in the susceptibility to GD. We aim to investigate the associations between HLA-DRB1 alleles and Taiwanese children with GD by both case-control and family-based studies. A total of 241 unrelated children with GD, 539 healthy controls, 115 trios of affected patients and their parents, and 121 trios of unaffected siblings and their parents were recruited. HLA-DRB1 genotyping was performed by polymerase chain reaction and sequence-based typing assays. We found that DRB1*09:01 (OR=2.60, 95% CI 2.02-3.35, Pc=6.55×10(-13)) was associated with GD risk, while DRB1*12:02 (OR=0.32, 95% CI 0.20-0.53, Pc=4.55×10(-5)) was protective against GD. Transmission/disequilibrium test further confirmed an overtransmission of the DRB1*09:01 (OR 3.37, 95% CI 2.13-6.22, Pc=1.0×10(-5)) and an undertransmission of the DRB1*12:02 (OR 0.21, 95% CI 0.05-0.42, Pc=1.7×10(-3)). The findings were similar in females when stratified by gender. In conclusion, our results clearly identify that HLA-DRB1*09:01 confers susceptibility to GD and DRB1*12:02 exerts protection against GD development in Taiwanese children.

Development of a high-throughput sequence-based typing assay for human leukocyte antigen loci

To develop a reliable assay for simultaneous sequence-based typing (SBT) of HLA-DPA1 and HLA-DPB1, and to apply it for the study of allelic polymorphisms in southern Chinese Han population. Based on full-length HLA-DPA1 and HLA-DPB1 allelic sequences, locus-specific PCR primers were designed and applied to amplify the target sequence encompassing the entire exon 2 of HLA-DPA1 and HLA-DPB1. PCR products were purified with magnetic beads, and run through an ABI 3730 DNA sequencer. Genotypes were assigned with an Assign 3.5 SBT software. The target sequences of HLA-DPA1 and HLA-DPB1 were both amplified with the PCR procedure. Little background and noise was observed in the derived sequences. Among 176 non-related healthy individuals, 4 HLA-DPA1 alleles with the frequencies of DPA1*02:02 (0.589) > DPA1*01:03 (0.284) > DPA1*02:01 (0.096) > DPA1*04:01 (0.031) were identified. In addition, 14 HLA-DPB1 alleles, including 4 common alleles (with a frequency of more than 5%, namely DPB1*05:01, DPB1*02:01, DPB1*04:01 and DPB1*02:02), 7 alleles with a frequency ranging from 1%-5% and 3 alleles with a frequency of less than 1% were identified. The results of HLA-DPB1 genotyping were all in accordance with the typing results derived from an Atria AlleleSEQR HLA-DPB1 kit. A reliable technique has been established for simultaneous genotyping of HLA-DPA1 and HLA-DPB1, which may have a broad application in population and disease association studies.

Molecular analysis of HLA-DRB1 allelic associations with systemic lupus erythematous and lupus nephritis in Taiwan

To evaluate the association of human leukocyte antigen (HLA)-DRB1 alleles with systemic lupus erythematosus (SLE) and lupus nephritis (LN) in the Taiwanese population, and to investigate the possible association of HLA-DRB1 alleles with disease severity in LN. HLA-DRB1 alleles were studied in 105 SLE patients (82 patients with LN, 23 patients without LN) and 855 healthy controls by polymerase chain reaction and sequence-based typing assays. The frequency of the HLA class II alleles DRB1*0301 (Odds ratio [OR] = 2.01, 95% confidence interval [CI] = 1.31-3.10, Pc = 0.02) and DRB1*1501 (OR = 2.06, 95% CI = 1.36-3.13, Pc = 0.01) were both increased in SLE patients, compared to healthy controls. The frequency of DRB1*1202 was significantly lower in LN patients than in SLE patients without nephritis (OR = 0.23, 95% CI = 0.09-0.57, Pc = 0.01). No specific allele was significantly associated with an increased or decreased risk for severity of LN in this sample. In Taiwanese people, the DRB1*0301 and DRB1*1501 alleles are significant risk factors for SLE, while the DRB1*1202 allele is protective for LN.

Leukocyte Antigen and Antibody Detection Assays: Tools for Assessing and Preventing Pulmonary Transfusion Reactions

Antibodies to neutrophil and HLA antigens can cause pulmonary transfusion reactions, and in some cases acute lung injury. When evaluating cases of pulmonary transfusion reactions, it is often necessary to test donors for neutrophil and HLA antibodies and also type the recipient for neutrophil and HLA antigens. A variety of enzyme-linked immunosorbent assay (ELISA) and flow cytometry-based solid phase assays are available to test for HLA class I and class II antibodies, but not neutrophil antibodies. Screening for neutrophil antibodies requires the preparation of panels of fresh neutrophils and testing in agglutination, immunofluorescence, or flow cytometry assays. Genotyping of HLA class I and II antigens is performed with a variety of sequence-specific primers, sequenced-specific oligonucleotide probe, and sequence-based typing assays. Neutrophil-specific antigens HNA-1a, -1b, -1c, -4a, and -5a can be genotyped, but not HNA-2a or -3a. Phenotyping of HNA-2a can be performed with CD177 monoclonal antibodies, but the gene encoding HNA-3a has not been identified, and the genomic basis for the HNA-2a-negative phenotype is not known. In conclusion, patients and donors involved with pulmonary transfusion reactions can be quickly typed for HLA antigens and tested for HLA antibodies, but testing for neutrophil antibodies and antigens requires the use of a reference laboratory.

Genetic Variations of HLA-DRB1 and Susceptibility to Kawasaki Disease in Taiwanese Children

Although some previous studies have reported that genetic and immunologic factors play important roles in the pathogenesis of Kawasaki disease (KD), the etiologic factors of this enigmatic pediatric disease are still poorly understood. The purpose of this study was to investigate whether polymorphisms of the human leukocyte antigen DRB1 (HLA-DRB1) gene are associated with KD and the development of coronary artery lesions (CAL) in Taiwanese children. Genomic DNA was extracted from whole blood samples from 145 children with KD and 331 healthy controls. The HLA-DRB1 gene was genotyped by polymerase chain reaction (PCR) and sequence-based typing assays. We found that the distribution of HLA-DRB1 allele families and alleles in children with KD did not differ from that in healthy controls. Stratified analysis did not demonstrate any association between particular HLA-DRB1 allele families or alleles and the development of CAL in children with KD. These findings suggest that susceptibility to KD and CAL is not associated with the HLA-DRB1 gene in a Taiwanese population. If immunogenetic determinants are involved in this disease and its complications in Taiwanese children, they must involve genes other than HLA-DRB1.

Evaluation of whole genome amplified DNA for use in HLA sequence based typing assays

Evaluation of whole genome amplified DNA for use in HLA sequence based typing assays