Characterization of the novel HLA-C*07:195 allele in an Italian hematopoietic stem cell volunteer donor, detected by sequence-based typing.

The new allele shows one nucleotide change from C*07:02:01:01 at 351 nt from C to A, resulting in an amino acid change at codon 93 of exon 3 from H to Q.

Resolution of HLA class I sequence-based typing ambiguities by group-specific sequencing primers.

The increasing demand for allele-level human leukocyte antigen (HLA) typing has led the sequence-based typing (SBT) to become the preferred method. In turn, the steady increase in the number of HLA alleles driven by the adoption of SBT as the ultimate typing method leads to the ever increasing number of cis/trans ambiguities. Over the last few years, additional sequencing with the commercially available group-specific sequencing primers (GSSPs) has replaced sequence-specific primer-polymerase chain reaction and group-specific amplification as the means of resolving cis/trans ambiguities in many laboratories. Here we summarize our 3-year experience in designing and utilizing GSSPs for resolution of HLA class I ambiguities. The panel of GSSPs used in our laboratory includes 14 primers for HLA-A, 18 for HLA-B, and 13 primers for HLA-C. The panel resolves 99.9% of all ambiguities.

Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification.

Molecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80.

A one-step real-time PCR assay for detection of DQA1*05, DQB1*02 and DQB1*0302 to aid diagnosis of celiac disease.

Celiac disease is an autoimmune disorder that develops after dietary exposure of the small intestine to gluten peptides in cereals. Celiac disease has a strong genetic component associated with HLA-DQ2 and HLA-DQ8, and testing for absence of these genetic markers is useful when serological tests and biopsies are indeterminate, as it renders celiac disease highly unlikely. We have developed a new real-time PCR assay, using sequence-specific primers (PCR-SSP) and TaqMan probes, for detection of DQB1*05, DQB1*02 (coding for DQ2) and DQB1*0302 (coding for DQ8). PCR amplification and detection of DQ2 and DQ8 was accurately and unambiguously performed from genomic DNA isolated from cell lines and human DNA. Amplification was scored digitally, without laboratory manipulation of amplified PCR products and with a higher accuracy than PCR-SSP. This assay should increase accuracy and throughput, and reduce risks of contamination in laboratories where testing for HLA DQ2 and DQ8 is performed as part of diagnosis of celiac disease.

Real-time PCR using fluorescent resonance emission transfer probes for HLA-B typing.

HLA genotyping by polymerase chain reaction (PCR) has some inherent labor-intensive and effort-demanding limitations. To overcome them, we have developed a real-time PCR with hybridization probes approach able to obtain a medium-low resolution HLA-B genotyping with fewer tubes and probes and with a shorter time requirement. Our strategy used 18 simultaneous reactions amplifying HLA-B alleles and an internal control. Monitorization of both amplifications in each tube is performed by the simultaneous application of two fluorescent resonance emission transfer probes: the first probe, different for each tube, is specific for the HLA-B locus and the second probe detects the control gene. A medium-low resolution (300 HLA-B allelic groups) typing is obtained for each sample by analyzing the melting curve patterns. Because some alleles may be determined without using the complete set of reactions, we present an alternative strategy: a first round of seven reactions and, according to the result, a second (or third) round of PCRs to solve the ambiguities. This method was validated in pretyped clinical samples and the results were completely concordant. Moreover, fewer ambiguous results were obtained. In summary, we present a new, faster, and more accurate method than currently used PCR techniques to type HLA-B alleles.

Validation of sensitive human leukocyte antigen-sequence-specific primer and probe typing in forensic DNA examination.

Validation studies were carried out with the commercially available HLA typing kit using a PCR-SPP (sequence-specific primer and probe) technique. This technique has made it possible to type class I (HLA-A and -B) and class II (HLA-DRB1 and -DQB1) alleles at low-resolution level with total 10 ng of template DNA, in addition to amplify directly from various forms of blood samples without DNA isolation procedure. Experimental examinations with bloodstains smeared on cotton cloth that were a week to 3 months old, bloodstains on gauze stored for 18 years, and buccal cells revealed that this HLA-SPP typing kit is a sensitive and reliable method for forensic investigations.

A method for using serum or plasma as a source of DNA for HLA typing.

A simple method for obtaining DNA from serum and plasma is described. Using appropriate primer pairs the polymorphic segments of HLA class II genes were amplified by the polymerase chain reaction (PCR) from this DNA, and typed using allele-specific oligonucleotide hybridization. When compared with DNA obtained from peripheral blood lymphocytes, the efficiency of the PCR was only minimally compromised and could be augmented by increasing the number of amplification cycles and/or by the addition of glycerol to the reaction mixture. This method serves as a reasonable alternative when no other source of DNA is available.

Extensive study of DRB, DQA, and DQB gene polymorphism in 23 DR2-positive, insulin-dependent diabetes mellitus patients.

To gain insight into the HLA subregions involved in protection against insulin-dependent diabetes mellitus (IDDM) we investigated the polymorphism of HLA-DR and -DQ genes in 23 DR2 IDDM patients. Results show the following. (1) Fourteen patients (61%) possess the DRB1, DRB5, and DQB1 alleles found in DRw16/DQw5 healthy people. These data contrast with the 5% of DRw16 normally found in DR2 populations and are in agreement with former observations supporting that the DRw16 haplotype is not protective. (2) Nine DR2 patients, i.e., 39% versus 95% in published DR2 controls, possess the DRB alleles found in DRw15 unaffected people. Among them, six patients have also DQA1 and DQB1 alleles identical to those found in DRw15/DQw6 healthy individuals. These data confirm that the DRw15/DQw6 haplotype is protective but indicate that none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRw15 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRw15. This strongly suggests that the occurrence of IDDM in these DRw15 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRw15/DQw6 haplotype. Finally, analysis of the non-DRw15 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQ alpha beta heterodimer of susceptibility.

A study of Italian pediatric celiac disease patients confirms that the primary HLA association is to the DQ(alpha 1*0501, beta 1*0201) heterodimer.

Celiac disease (CD) has been recently reported to be primarily associated with the DQ(alpha 1*0501, beta 1*0201) heterodimer encoded in cis on DR3 haplotype and in trans in DR5,7 heterozygous individuals. The high incidence of DR5,7 heterozygotes, reflecting the high frequency of the DR5 allele in Italy, makes the analysis of the Italian CD patients critical. Polymerase chain reaction-amplified DNA from 50 CD patients and 50 controls, serologically typed for DR and DQw antigens, was hybridized with five DQA1-specific oligonucleotide probes detecting DQA1*0101 + 0102 + 0103, DQA1*0201, DQA1*0301 + 0302, DQA1*0401 + 0501 + 0601, and DQA1*0501 and a DQB1-sequence-specific oligonucleotide probe recognizing DQB1*0201 allele. As expected by the DR-DQ disequilibria, DQA1*0201 [62% in patients versus 26% in controls, relative risk (RR) = 5] and DQA1*0501 (96% versus 56%, RR = 19) show positive association with the disease. Of CD patients, 92% (50% DR3 and 42% DR5,7) compared to 18% of the controls carry both DQA1*0501 and DQB1*0201 alleles, so that the combination confers an RR of 52, higher than both the risks of the single alleles (DQA1*0501 RR = 19, DQB1*0201 RR = 30), confirming the primary role of the dimer in determining genetic predisposition to CD both in DR3 and in DR5,7 subjects.

Identification of three novel HLA class I alleles: HLA-B*3928, HLA-B*400104 and HLA-B*4437.

Three new human leukocyte antigen (HLA) class I alleles have been identified in the Tissue Typing Laboratory in Sydney, Australia. Sequence analysis of exon 2 and exon 3 of the HLA-B gene revealed the novel polymorphism. A silent substitution of C to T at nucleotide position 369 has been identified for the HLA-B*400104 allele when compared to the closest matched allele, HLA-B*400101. The HLA-B*3928 allele was identified with a nucleotide substitution of G to C at position 362 when compared to the closest matched allele, HLA-B*390101, resulting in an amino acid substitution of Arginine to Threonine. A nucleotide substitution of C to G at position 572 resulting in the amino acid change Serine to Tryptophan was identified in the new allele HLA-B*4437, when compared to the closest matched allele HLA-B*440301. Both amino acid substitutions implicate a different specificity and affinity of antigen binding for the alleles HLA-B*3928 and HLA-B*4437.

PCR-SSOP molecular typing of HLA-C alleles in an Iranian population.

HLA-C alleles were characterized by a polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) hybridization protocol in a sample of 120 Iranians from Tehran. A total of 23 alleles were identified with the four most predominant--Cw*0401, Cw*0602, Cw*1202, and Cw*0701/06--accounting for almost 50% of HLA-C alleles. A comparison of HLA-C diversity among several populations indicates that Iranians stand at an intermediate genetic position between Europeans and Africans, an observation that may be related to their geographical location at a continental crossroads. The results also reveal a very high correlation between genetic and geographic distances on a global scale. A total of 30 HLA-C-DRB1 haplotypes were found in the Iranians, with the highest frequencies of 6.6% and 6.04 % being for Cw*0602-DRB1*0701 and Cw*1202-DRB1*1502, respectively.

Ambiguities of human leukocyte antigen-B resolved by sequence-based typing of exons 1, 4, and 5.

The elucidation of the sequences of human leukocyte antigen-B (HLA-B)-exons 1 through 5 has led to an increase of ambiguities with alleles having identical exon 2 and 3 sequences, but differences in other exons. At the moment, 26 HLA-B alleles show such ambiguities which can be resolved by sequencing the exons in which the differences are located. Here we report a sequence-based typing (SBT) strategy for heterozygous sequencing of exons 1, 4, and 5, in addition to the previously described exons 2 and 3. The strategy was validated against a panel of 25 individuals, carrying HLA-B alleles from 33 different allele groups. Correct assignment of all HLA-B alleles was obtained for exons 1 through 5. In addition, the SBT protocol was used to resolve ambiguities in 50 individuals. The ambiguous combinations studied were B*0705/06, B*0801/19N, B*1512/19, B*180101/17N, B*270502/13/0504, B*350101/42/40N, B*390101/0103, B*400102/0101, B*440201/19N/27, and B*510101/11N/0105/30/32. In all cases, sequencing revealed the first allele to be present, except for three individuals with B*07. One of them typed B*0705; the other two were B*0706. The described SBT protocol for sequencing exons 1, 4, and 5 is a valuable tool for resolving ambiguities of HLA-B alleles with differences in these exons, as well as for studying the polymorphism of HLA-B outside exons 2 and 3.

Identification of DRB alleles in rhesus monkeys using polymerase chain reaction-sequence-specific primers (PCR-SSP) amplification.

Major histocompatibility complex (MHC) class In molecules play a vital role in the regulation of T-cell functions in the mammalian immune system. Two key features characterize the polymorphism of MHC haplotypes in humans and non-human primates: the existence of a large number of alleles, and the high degree of genetic diversity between those alleles. Rhesus monkeys and Chimpanzees have been extensively used as relevant models for human diseases and transplantation We have investigated DRB genes in 19 macaques, members of 3 families, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and denaturing gradient gel electrophoresis (DGGE). After amplification PCR products were purified and subjected direct sequencing. Seven animals (Madison #1) were typed by DDGE also. We report that the DRB haplotypes defined by PCR-SSP exhibit a high degree of concordance with the data obtained by DGGE and direct sequening. Our data show prominent variability in the number of DRB1 alleles ranging from 1-4 per genotype within these families. This analysis demonstrated that most of the amplicons were identical to Mamu-DRB alleles that our PCR primers were to amplify. However, 98-99% similarity was noticed in the case of Mamu-DRB1*0303, Mamu-DRB6*0103 and Mamu-DRB*W201 alleles. The observed mismatches were located in non-polymorphic regions. Thus, family studies in rhesus macaques performed by molecular methods confirmed the multiplicity of Mamu-DRB1 alleles per haplotype and the existence of allelic associations published earlier. In addition, we propose 3 more DRB allele associations (haplotypes): Mamu-DRB1*04-DRB5*03; Mamu-DRB1*04-*DRB*W5; Mamu-DRB1*04*W2. The proposed medium-resolution PCR-SSP technique appears to be a highly reproducible and discriminatory typing method for detecting polymorphisms of DRB genes in rhesus monkeys.

HLA-DR polymorphism in a Senegalese Mandenka population: DNA oligotyping and population genetics of DRB1 specificities.

HLA class II loci are useful markers in human population genetics, because they are extremely variable and because new molecular techniques allow large-scale analysis of DNA allele frequencies. Direct DNA typing by hybridization with sequence-specific oligonucleotide probes (HLA oligotyping) after enzymatic in vitro PCR amplification detects HLA allelic polymorphisms for all class II loci. A detailed HLA-DR oligotyping analysis of 191 individuals from a geographically, culturally, and genetically well-defined western African population, the Mandenkalu, reveals a high degree of polymorphism, with at least 24 alleles and a heterozygosity level of .884 for the DRB1 locus. The allele DRB1*1304, defined by DNA sequencing of the DRB1 first-domain exon, is the most frequent allele (27.1%). It accounts for an unusually high DR13 frequency, which is nevertheless within the neutral frequency range. The next most frequent specificities are DR11, DR3, and DR8. Among DRB3-encoded alleles, DR52b (DRB3*02) represents as much as 80.7% of all DR52 haplotypes. A survey of HLA-DR specificities in populations from different continents shows a significant positive correlation between genetic and geographic differentiation patterns. A homozygosity test for selective neutrality of DR specificities is not significant for the Mandenka population but is rejected for 20 of 24 populations. Observed high heterozygosity levels in tested populations are compatible with an overdominant model with a small selective advantage for heterozygotes.

DPB1 polymorphism in rheumatoid arthritis: evidence of an association with allele DPB1 0401.

HLA-DP polymorphism was examined in 71 rheumatoid arthritis patients and 148 controls, using dot-blot analysis with 14 synthetic oligonucleotide probes specific for the variable region of the DPB1 second exon. The DPB1 0401 allele was found to be significantly more frequent in RA patients than in controls (77.46% vs 55.40%, p less than 0.002, pc less than 0.03, relative risk value: 2.74). An association between DPB1 0401 and seropositivity for rhumatoid factors was also observed: 44 of the 55 seropositive RA patients were DPB1 0401 (p less than 0.001). Analyzing the HLA DPB1 alleles frequencies in 57 HLA-DR-typed RA patients did not show any linkage between the DPB1 0401 and the DR4 specificities. Furthermore, the DPB1 0401 homozygous frequency was increased in DR4-negative RA patients. Our findings suggest an independent role of the DPB1 0401 allele in the genetic susceptibility to RA.

Alloantisera can detect some of the variants of HLA DRw13 identified by oligotyping.

Oligotyping has revealed a considerable polymorphism of HLA DRw13; so far, 5 alleles coded by DRB1 have been identified. An even greater number of haplotypes are apparent when considering the association of DRw13 with alleles coded by DRB3 and DQB1. Serology can now define some of these variants which had formerly escaped detection. We have tested by serology and by oligoprobes 66 genotyped Caucasoid cells comprising the known DR and DQ alleles. Among the 28 DRw6 cells, 10 different haplotype patterns were selected, of which 8 concern DRw13 and 2 DRw14. Four variants of DRw13 were represented: DRB1 *1301, *1302, *1303 and *1305 associated with usual and with uncommon alleles belonging to DRB3 and to DQB1. We have centered our analysis on 19 sera of the XIth Workshop defining DRw6. Two sera (639, 826) were noteworthy since they reacted with certain subtypes of DRw13 and with DRB1 *1102, *1103 only, the other DR specificities remaining negative.

Oligotyping of Italian celiac patients with the 11th International Histocompatibility Workshop reagents.

Oligotyping for HLA-DRB1, DQA1 and DQB1 specificities has been performed on PCR-amplified DNA from 55 Italian celiac children, living in Rome, and 50 blood donors. 52.6% of CD patients were DR3;DQA1*0501;DQB1*0201-positive versus 14% of controls (RR = 6.85) and 34.5% were DR5,7;DQA1*DQB1*0201-positive versus 2% of controls (RR = 25.86). 7 patients (12.7%) were negative for the DQA1*0501/B1*0201 dimer: 3 of them were DR4 (5.4%) and the others typed as DR1,5; 1,7; 5,7 and w6,7. No patient was negative for both DQA1*0501 and DQB1*0201 alleles.

Polymorphism of HLA in the Romanian population.

We have investigated the HLA-class I and class II polymorphism in a population of 83 Romanians using conventional serology together with PCR amplification and oligonucleotide typing of HLA-class II genes. Romanians show a higher frequency of HLA-A11, B13, B18, B37, B39, B51 and DR2 than other European populations. HLA-DRB1*1501 and 1601 account for the high frequency of the serologic specificity DR2. In Romanians, HLA-DR2 is in linkage disequilibrium with HLA-B18 and HLA-Bw52 rather than with HLA-B7 as in the case in other Europeans. Unexpected HLA-DR2 haplotypes include HLA-DRB1*1502, DQA1*0102, DQB1*0601; HLA-DRB1*1602, DQA1*0102, DQB1*0502. Other unusual haplotypes include HLA-DRB1*0405, DQA1*03, DQB1*0302; HLA-DRB1*1305, DQA1*0103, DQB1*0603; and HLA-DRB1*1405, DQA1*0101, DQB1*05032. Analysis of the genetic distance between Romanians and other Europeans who have been studied serologically are consistent with the hypothesis that Romanians descend from Roman ancestors who colonized Dacia between the 1st century B.C. and 1st century A.D.