Sequence-specific Oligonucleotide Probe Typing Research Articles

Analysis and verification of a HLA-DQB1*03:90N allele with a single base deletion

To verify a HLA-DQB1*03:90N allele and method to improve the accuracy of HLA typing. A total of 2265 hematopoietic stem cell donors from Shenzhen Branch of China Marrow Donor Program in 2018 were initially detected by a PCR sequence-specific oligonucleotide probe (SSOP) method. Among these, a rare HLA-DQB1 allele was identified by sequence-based tying (SBT) and Ion Torrent S5 next generation sequencing (NGS). The SSOP typing result suggested the HLA-DQB1 to be a rare allele, while an insertion and a deletion was suspected in its exon 2 by SBT, which were confirmed by NGS as DQB1*03:90N and DQB1*06:01, respectively. Rare alleles suspected by the SSOP method should be verified by other methods to ensure the accuracy of HLA genotyping. Rare alleles formed by deletions can be detected by NGS with accuracy.

Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design

HLA-B*57:01 genotyping before abacavir (ABC) administration is a standard of care to avoid ABC-driven hypersensitivity reactions. Several HLA-B*57:01 tests have been developed, each with advantages and disadvantages. Some have limited accuracy, require special instrumentation, and/or are labor intensive and expensive. We developed a novel hydrolysis probe-based real-time PCR method of HLA-B*57:01 genotyping. Primer and probes were designed based on published sequence variations in exon 3 of HLA-B that distinguish HLA-B*57:01 from ABC-insensitive alleles such as HLA-B*57:03 and HLA-B*58:01. We designed PCR primers to amplify HLA-B*57:01 along with closely related alleles, such as HLA-B*57:03, directly from genomic DNA. Most ABC-insensitive alleles, including HLA-B*58:01, would not produce any products in the PCR reaction. Our hydrolysis probes enable differentiation of HLA-B*57:01 from the other amplified, but ABC-insensitive, alleles. In addition to using real-time PCR, we used restriction enzymes to generate differential digestion patterns that led to the development of an HLA-B*57:01 PCR-restriction fragment length polymorphism marker. When used to genotype a set of 75 selected clinical samples, our real-time PCR assay demonstrated 100% accuracy in distinguishing between the HLA-B*57:01-positive and -negative alleles when results were compared to those of sequence-specific oligonucleotide probe typing and reference laboratory testing. Our newly developed test will allow clinical laboratories with real-time PCR capabilities to perform HLA-B*57:01 genotyping in a timely and economical manner.

HLA-A allele dropout in sequence-specific oligonucleotide probe typing due to intronic polymorphism in the novel A*31:01:02:02 allele.

HLA-A*31:01:02:02 differs from A*31:01:02 in a single nucleotide mutation at intron 3, nucleotide position 1000 (G > A).

Stratifying Risk for Celiac Disease in a Large At-Risk United States Population by Using HLA Alleles

Susceptibility to celiac disease (CD) is related to HLA-DQ2 and DQ8 alleles and the heterodimers they encode. The objective of this study was to stratify risk for CD on the basis of HLA-DQ genotype. DNA from 10,191 subjects who are at risk for CD was analyzed for HLA-DQ haplotypes. Individuals with CD were identified as those who tested positive for anti-endomysial immunoglobulin A (EMA+) in an immunofluorescence assay. Samples homozygous for DQ2.5 (HLA-DQA1 05-DQB1 02) or DQ2.2/DQ2.5 (HLA-DQA1 05-DQB1 02 and HLA-DQA1 0201-DQB1 02) comprised 5.38% of the total; 28.28% of these were EMA+ (95% confidence interval [CI], 24.55-32.26). Of the samples that were DQ2.5 heterozygous (HLA-DQA1 05-DQB1 02); 9.09% were EMA+ (95% CI, 7.82-10.51). Among samples in which HLA-DQ8 (HLA-DQA1 03-DQB1 0302) was detected, 8.42% of homozygotes (95% CI, 3.71-15.92) and 2.11% of heterozygotes (95% CI, 1.43-3.00) were EMA+. Samples with DQ2.2/DQ8 or DQ2.5/DQ8 comprised 5.08% of the total, and 11.78% of these were EMA+ (95% CI, 9.13-14.87). HLA-DQ2 and HLA-DQ8 were absent in 4283 samples (42.03% of the total); 0.16% of these samples were EMA+ (95% CI, 0.07-0.34). High-resolution, sequence-specific oligonucleotide probe typing with 35 DQA1-specific and 37 DQB1-specific probes of DNA from more than 10,000 subjects was used to stratify risk of CD in an at-risk U.S. population. DQ2 homozygosity (DQ2.5/DQ2.2+2.5) increased risk for CD, estimated by the rate of EMA positivity, compared with the entire sample population and other DQ genotypes. These data suggest a quantitative relationship between the type/proportion of DQ heterodimers and the risk of CD and identify potential immunotherapeutic targets.

Sensitivity to Bee Venom Antigen Phospholipase A 2: Association With Specific HLA Class I and Class II Alleles and Haplotypes in Beekeepers and Allergic Patients

Bee venom hypersensitivity is a clinical entity of outstanding importance because bee stings are a leading cause of mortality worldwide. Individuals with immediate-type bee venom hypersensitivity, beekeepers, and healthy controls were examined for HLA-DRB1, DQB1, and DQA1 alleles by sequence-specific oligonucleotide probe typing. Defined hypersensitivity to bee venom antigen phospholipase A 2 (vbPLA 2) is significantly associated with the presence of susceptible HLA class II alleles: DRB1*0101 (RR = 2.7, p < 3 × 10 −3), DRB1*0103 (RR = 21.2, p < 7.5 × 10 −11), DQA1*0101 (RR = 1.2, p < 38.52 × 10 −10), and DQB1*0501 (RR = 4, p < 2.18 × 10 −10). Some HLA class I alleles were also associated with risk to bee venom allergy: A*01 (RR = 2.4, p < 7.5 × 10 −4), B*57 (RR = 35.1, p < 3.5 × 10 −7), and B*5901 ( p < 3.5 × 10 −5), but they are probably of secondary significance. Three- (DRB1*0103-DQA1*0101-DQB1*0501) (RR = 21.24, p < 7.5 × 10 −11) and five-loci (A*01-B*59-DRB1*0103-DQA1*0101-DQB1*0501) ( p < 2.3 × 10 −6) extended haplotypes are also significantly carried by vbPLA 2 allergic patients. When HLA allele frequencies from patients are compared with those from beekeepers, only HLA-DRB1*0103 (RR = 11.7, p < 8.5 × 10 −5) and HLA-DQA1*0101 ( p < 0.02) were significantly increased in the former. These observations emphasize the importance of the DRB1*0103-DQA1*0101-DQB1*0501 haplotype as a strong candidate for susceptibility to vbPLA 2 hypersensitivity, at least in our region.

Investigation of killer cell immunoglobulin-like receptor gene diversity: II. KIR2DS4

A polymerase chain reaction–sequence-specific oligonucleotide probe typing method identifying and distinguishing alleles of the KIR2DS4 gene has been established. The system is based on the specific amplification of a region of this gene, followed by hybridization with 11 sequence-specific oligonucleotide probes. The method has been applied to a healthy group of Northern Irish caucasian individuals, establishing frequencies of alleles of this locus within the local population. Furthermore, cell line DNA and families from the 13th International Histocompatibility Workshop, in addition to local families, have also been allele typed at the KIR2DS4 locus. Haplotype segregation, with respect to KIR2DS4 alleles, has been examined by using the local family data. Within all sample groups investigated, four KIR2DS4 alleles were identified, two of which are novel to this investigation.

Association of hypersensitivity to the nematode Anisakis simplex with HLA class II DRB1∗1502-DQB1∗0601 haplotype

Anisakiasis as well as allergic and anaphylactoid reactions to Anisakis simplex antigens are recently identified clinical entities. They are relatively frequent in countries with habitual raw food consumption, often in the form of large amounts of fish and sea food products. In this communication the relationship between HLA class II alleles and the IgE-specific immune response to A. simplex allergen was studied in a defined population in Northern Spain. Individuals with immediate-type Anisakis hypersensitivity and healthy controls were examined for HLA-DRB1, DQB1 and DQA1 alleles by sequence-specific oligonucleotide probe typing. Analysis of the HLA data among patients revealed increased phenotypic frequencies for DRB1∗1502 and DRB1∗0404 compared to healthy controls ( p < 1 × 10 −7 and < 0.01, respectively). Analysis of haplotypic frequencies showed that the DRB1∗1502-DQB1∗0601 haplotype is significantly higher in patients with Anisakis hypersensitivity in comparison with the control population from the same region ( p < 4 × 10 −8). The data suggest that this haplotype can be considered to be a susceptibility factor for hypersensitivity to A. simplex antigens.

HLA class II antigens DR4 and DQ8 are associated with allergy to hevein, a major allergen of Hevea latex.

In this study we investigated the relationship between HLA class II alleles and the IgE-specific immune response to the 4.7 kDa polypeptide hevein of Hevea brasiliensis, a major latex allergen, 51 individuals with immediate-type latex allergy and 90 controls were examined for the polymorphisms in exon 2 of HLA-DRB1, 3, 4, 5 and DQB1 by sequence-specific oligonucleotide probe typing. 35 (69%) out of 51 latex-sensitized subjects showed positive hevein-specific IgE values. Analysis of the HLA data among these 35 subjects revealed increased phenotype frequencies for DR4 (22/35, 63%) and DQ8 (18/35, 51%) when compared with those in the 16 hevein-negative but latex-positive subjects (DR4: 2/16, 13%, p = 0.0009, Pc = 0.047; DQ8: 0/16, p = 0.0003, pc = 0.018) and with healthy controls (DR4: 22/90, 24%, p = 0.00012, pc = 0.013; DQ8: 16/89, 18%, p = 0.0003, pc = 0.036). Finally the DR4-DQ8 haplotype frequency was significantly elevated in hevein-positives when compared with hevein-negatives (51% vs. 0, p = 0.0003, pc = 0.034) or controls (51% vs. 18%, p = 0.0002, pc = 0.045) The present data suggest DR4 and DQ8 to be operating jointly as susceptibility factor for the allergy to hevein.

DNA typing of cadaveric donors by a rapid sequence specific oligonucleotide probe typing kit

DNA typing of cadaveric donors by a rapid sequence specific oligonucleotide probe typing kit

Combinatorial diversity in DR2 haplotypes.

Sequence analysis has identified multiple alleles at two loci that encode for the DR2 specificity. The loci, DRB1 and DRB5, are in linkage disequilibrium which can extend to alleles of the DQ loci. Serologic, cellular, and sequence-specific oligonucleotide probe (SSOP) typing techniques have been used to identify the DR2 haplotypes. In this report, we have characterized by SSOP typing and cDNA/DNA sequence analyses the combinatorial diversity of DR2 haplotypes. Cells were selected on the basis of unique serologic reactivity, unique associations of alleles of DR and DQ loci, and/or presence in populations which have not been extensively characterized for HLA diversity. An asymmetric polymerase chain reaction (PCR) amplification was applied to rapidly screen unique cells and to characterize DNA sequence in conjunction with more conventional cDNA sequence analysis. The sequence data confirm the lack of a DRB5 locus in the DR2"LUM" specificity, the unexpected association of DRB1*1602 and DRB5*010 alleles in a nonCaucasoid population, and the association of the allele DRB1*1503 with DRB5*0101 in black African, African American and native American individuals. The DRB1*1503 and DRB5*0101 alleles were identified in an unusual haplotype, DR2,DQ2. The combinatorial diversity of the DR2 haplotypes is extended by these studies in nonCaucasoid populations.

Polymorphism of HLA‐DRw52‐associated DRB1 genes as defined by sequence‐specific oligonucleotide probe hybridization and sequencing

We have used group-specific DNA amplification and sequence-specific oligonucleotide probe (SSOP) hybridization to study DRB1 sequence polymorphisms associated with DR3, DRw11(5), DRw12(5), DRw13(w6), DRw14(w6) and DRw8 alleles. Group-specific amplification of DRw52-associated DRB1 alleles was achieved using a 5' amplification primer designed to hybridize with a first hypervariable region (HVR) sequence common to all known alleles in this group, together with a 3' intron primer. Prospective SSOP typing of DR3, DRw11, DRw12, DRw13, DRw14 and DRw8 alleles was performed in 318 individuals, including 124 patients, 46 family members and 148 unrelated marrow donors. Among the 395 DRw52-associated DRB1 alleles tested in our study, a subtype corresponding to the previously defined alleles DRB1*0301-2 (DR3), DRB1*1101-4 (DR5), DRB1*1201-2 (DR5), DRB1*1301-5 (DRw6), DRB1*1401-2 and 1404 (DRw6), and DRB1*0801-4 (DRw8) could be assigned in all but 6 individuals (1.9%) tested. In addition to the 22 known alleles, we identified two new DRw6-associated alleles, DRB1*13.MW(1) and DRB1*14.GB(1). DRB1*13.MW typed serologically as DRw13 and was identical to DRB1*1301 except at codon 71 where AGG encodes arginine instead of GAG encoding glutamic acid. DRB1*14.GB represents a DRB1*1402 variant whose sequence at codon 86 encodes valine (GTG) instead of glycine (GGT). These results demonstrate that SSOP methods represent an efficient and precise approach for typing DRB1 alleles and for identifying potential novel variants previously unrecognized by conventional typing methods.

Ten HLA-DR4 alleles defined by sequence polymorphisms within the DRB1 first domain

We have studied DRB1 sequence polymorphisms associated with DR4 subtypes using DR4-specific DNA amplification and sequence-specific oligonucleotide probe (SSOP) hybridization. The 5' amplification primer was designed to hybridize with a unique sequence in the first hypervariable region (HVR) of the DRB1 second exon of all known DR4 alleles; the 3' primer was designed to hybridize with an intron sequence common to all DRB1 The specificity of the amplification step was demonstrated by double-blind testing of 105 selected DNA samples. Prospective SSOP typing of DR4 alleles was performed in 104 unrelated individuals known to be DR4-positive, including 13 who were DR4-homozygous. A DRB1 subtype corresponding with the previously defined DR4-associated specificities Dw4, Dw10, Dw13.1, Dw13.2, Dw14.1, Dw14.2, Dw15, and DwKT2 could be assigned for each of the 117 DR4 haplotypes tested. In most cases, DR4-homozygous, DRB1-heterozygous individuals could be genotyped with the panel of probes. In the course of our analysis, we identified two new DR4-related alleles, DRB1*04.CB (DRB1*0410)1 and DRB1*04.EC (DRB1*, 0411)2 which were recognized by their novel hybridization patterns. The DRB1 second exon sequence of DRB1*04.CB is identical to DRB1*0405 except at codon 86 where GTG encodes valine instead of GGT encoding glycine. DRB1*04.EC is identical to DRB1*04.CB except at codon 74 where GAG encodes glutamic acid instead of GCG encoding alanine. Our results provide further evidence that SSOP hybridization is the most effective approach available for subtyping DR4 haplotypes and identifying unrecognized variants. A similar approach should be equally informative for subtyping other DR alleles.