bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunogenetic study of sympathetic ophthalmia
Authors: Shindo Y. , Ohno S. , Usui M. , Ideta H. , Harada K. , Masuda H. , Inoko H.
Published: December 2008

Sympathetic ophthalmia (SO) is very rare but it remains one of the most intractable eye diseases. In clinical manifestations and histopathologic features SO is known to resemble closely Vogt‐Koyanagi‐Harada's disease (VKH disease). We had reported that VKH disease was significantly associated with HLA‐DRB1*04 and ‐DQB1*04 in Japanese patients. In this study, to investigate an HLA association with SO we performed HLA serological and PCR‐based DNA typing in 16 patients and 50 healthy controls. Our study revealed that HLA‐DRB1*04 (0405; Pc<5×10A DQA1*03 (Pc<5×10‐3), and DQB1*04 (0401; Pc<5times10‐4) were significantly associated with SO as compared to the healthy controls but there was no significant difference in the frequencies of any DPB1 alleles between the patients and healthy controls. It can be postulated that not only the clinical manifestations but also the genetic predisposition of SO are very similar to those of VKH disease.
High polymorphism of Mhc‐E locus in non‐human primates: alleles with identical exon 2 and 3 are found in two different species
Authors: Alvarez M. , Martinez‐Laso J. , Varela P. , Diaz‐Campos N. , Gomez‐Casado E. , Vargas‐Alarcon G. , Garcia‐Torre C. , Amaiz‐Villena A.
Published: December 2008

Thirteen Mhc‐E new sequences were found in eight individuals belonging to the Cercopithecinae family, i.e.: Macaca mulatta, Macaca fascicularis and Cercopithecus aethiops when studying E locus polymorphism. No changes were found in the invariant residues which are required for the correct conformation of the peptide presenting region which are conserved in classical Mhc class I molecules from fish and reptiles to humans; however, polymorphism of Mhc‐E alleles is not limited to the three typical hypervariable regions per domain as it is in classical class I alleles. The rate of synonymous and non‐synonymous substitutions in the DNA sequence corresponding to the antigen binding site, compared to the remainder ofexons 2 and 3 shows that the peptide‐binding site is under high evolutionary pressure for stability since only synonymous substitutions have been found to be accepted in apes. Also, a clear example of trans‐species evolution of allelism is found: two identical exon 2 and exon 3 sequences there exist belonging to individuals from different species (Mamu‐Mhc‐E*0101 and Mafa‐Mhc‐E*04). In addition, two Macaca mulatta individuals show an Mhc‐E locus duplication. Finally, phylogenetic tree analysis shows that Mhc class I molecules found in Saguinus oedipus (described as Mhc‐G homologues) are closer to Mhc‐E sequences.
Transporter associated with antigen‐processing (TAP) genes and susceptibility to tuberculoid leprosy and pulmonary tuberculosis
Authors: Rajalingam R. , Singal D. P. , Mehra N. K.
Published: December 2008

We have studied TAP polymorphism in a panel of 40 healthy individuals, 57 patients with pulmonary tuberculosis (PTB) and 50 with tuberculoid (TT) leprosy from North India. Only TAP2‐A/F occurred with a significantly increased frequency in PTB patients as compared to controls (82.5% vs. 52.5%, P<0.002, Pc<0.01) giving a high relative risk of 4.3. On the other hand, TAP2‐B was significantly increased in TT leprosy as compared to controls (76% vs. 47.5%, Pc<0.003, RR 3.5) particularly in patients positive for HLA‐DR15 than controls carrying DR15 (77.5% vs. 50%, P<0.03, RR=3.4). Further, TAP2‐B allele was positively associated with DR15 negative PTB patients as compared to the DR15 positive group (43.8% vs. 17.1%, P<0.04, RR=0.3), This study along with our earlier studies on HLA association in mycobacterial diseases suggests that in addition to HLA‐DR15, alleles in the TAP2 region influence susceptibility to PTB and TT leprosy.
TAP polymorphisms in Swedish myasthenia gravis patients
Authors: Hjelrnström P. , Giscombe R. , Lefvert A. K. , Pirskanen R. , Kockum I. , Landin‐Olsson M. , Sanjeevi C. B.
Published: December 2008

The association between myasthenia gravis (MG) and TAP polymorphisms was studied in 79 Swedish patients and 155 unrelated controls. TAP typing was performed by ARMS‐PCR technique and stratification analysis was used to determine if the TAP associations were independent or secondary to linkage disequilibrium with DQ2 and DR3. TAP1 and TAP2 alleles did not confer independent risk for MG. TAP2*0101 was, however, positively associated with MG in patients with an early onset of disease compared to patients with a late onset of disease. TAP1 and TAP2 alleles did not confer risk in MG patients negative for DQ2. In conclusion, susceptibility to MG is not primarily conferred by TAP alleles in the extended DR3 haplotype.
A novel DRB1*13 allele (DRB1*1327) on a DR17, DQ2 haplotype with a DRB1*0301 sequence motif in the 2nd hyperpolymorphic region
Authors: Schaffer M. , Olerup O.
Published: December 2008

In a sample from a Netherlands Caucasian, we found a new DRB1*13 allele (DRB1*1327). The nucleotide sequence of the second exon of the novel allele was identical to DRB1*1301 except for a single productive base substitution changing codon 26 from TTC to TAC, encoding phenylalanine and tyrosine, respectively. The new allele shares sequence with DRB1*03011 from codons 5 to 66. The haplotype carrying the new allele was, from known linkage disequilibria, deduced to be DRB1*1327, DRB3*0101, DQA1*05011, DQB1*0201, i.e. similar to the DR17, DQ2 haplotype, which suggests that the DRB1*1327 allele has arisen by a double recombination event between a DR13 donor haplotype and a DR17, DQ2 recipient haplotype.
HLA‐B*27 typing by PCR—restriction fragment length polymorphism
Authors: Nieto A. , Fraile A. , Vinasco J. , Martín J.
Published: December 2008

In this study we describe the use of PCR‐RFLP for genotyping HLA‐B*27. A 557 bp fragment from HLA‐B locus is amplified and subjected to digestion with StyI. The presence of B*27 is detected on electrophoresis by the appearance of 431+126 bp pattern. The same pattern could be obtained only for the very infrequent allele B*7301. However, this allele was not amplified in the B73 sample tested with the primers and conditions used in this study. Nevertheless, we have designed two PCR‐RFLP approaches for separating these alleles. The PCR‐RFLP method was tested on a panel of forty‐three cell lines and applied to fifty spondyloarthritic patients and one‐hundred‐eighty healthy subjects. Given its robustness, technical simplicity and cost‐effectiveness, we think that this method can be incorporated for routine use in most laboratories
Extensive HLA class II studies in 58 non‐DRB1*15 (DR2) narcoleptic patients with cataplexy
Authors: Mignot E. , Kimura A. , Lattermann A. , Lin X. , Yasunaga S. , Mueller‐Eckhardt G. , Rattazzi C. , Lin L. , Guilleminault C. , Grumet F. C. , Mayer G. , Dement W. C. , Underhill P.
Published: December 2008

Narcolepsy is a sleep disorder that has been shown to be tightly associated with HLA DR15 (DR2). In this study, 58 non‐DR15 patients with narco‐lepsy‐cataplexy were typed at the HLA DRB1, DQA1 and DQB1 loci. Subjects included both sporadic cases and narcoleptic probands from multiplex families. Additional markers studied in the class II region were the promoters of the DQA1 and DQB1 genes, two CA repeat polymorphisms (DQCAR and DQCARII) located between the DQA1 and DQB1 genes, three CA repeat markers (G51152, T16CAR and G411624R) located between DQB1 and DQB3 and polymorphisms at the DQB2 locus. Twenty‐one (36%) of these 58 non‐DR15 narcoleptic patients were DQA1*0T02 and DQBI*0602, a DQ1 Subtype normally associated with DRB1*15 in DR2‐positive narcoleptic subjects. Additional microsatellite and DQA1 promoter diversity was found in some of these non‐DR15 but DQB1*0602‐positive haplotypes but the known allele specific codons of DQA1*0102 and DQB1*0602 were maintained in all 21 cases. The 37 non‐DQA1*0102/DQB1*0602 subjects did not share any particular HLA DR or DQ alleles. We conclude that HLA DQA1*0102 and DQB1*0602 are the most likely primary candidate susceptibility genes for narcolepsy in the HLA class II region.
The novel HLA‐DRB1*0807 allele is present in a Carib Amerindian population: the Yucpa
Authors: Layrisse Z. , Matos M. , Montagnani S. , Herrera F.
Published: December 2008

Nucleotide sequences of five novel HLA‐DRB1*08 alleles have been recently reported but their frequency in different populations is still unknown. The presence of DRB1*08 variants among Carib‐speaking Yucpa Amerindians of the Perijá Range in the border between Colombia and Venezuela was tested by PCR‐DNA typing. DRB1*0807 was the only DRB1*08 variant found in this population with a frequency of 0.1250. Until very recently, DRB1*0802 was the predominant DRB1*08 allele present among full‐blooded Amerindians, I but in the last 3 years DRB1*0804 has been found in 2 South American tribes and *0811 in 2 North American tribes. The description of a new functional DRB1*08 variant in Amerindians suggests that extensive gene polymorphism among Amerindians might not be limited to the HLA‐B Class I locus.
Molecular genetic analysis of HLA class II alleles in Japanese patients with melanoma
Authors: Kageshila T. , Naruse T. , Hiral S. , Ono T. , Horikoshi T. , Nakagawa H. , Tamaki K. , Hayashibe K. , Ichihashi M. , Nakayama J. , Hori Y. , Ozawa A. , Miyahara M. , Ohkido M. , Inoko H.
Published: December 2008

Distribution of HLA‐DQA, ‐DQB and ‐DPB alleles in ninety‐six Japanese patients with melanoma was analyzed using the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method, and the association between clinical parameters and the presence of certain HLA class II alleles investigated. The frequency of HLA‐DQB 1*0302 was increased, while those of DQA1*0101(04), ‐DQA1*0401 and DRB1*0802 were decreased in melanoma patients compared with controls. Moreover, the frequency of HLA‐DQA 1*0103 in patients with acral lentiginous melanoma was increased compared with controls. However, none of these HLA class II alleles showed significant positive or negative associations after correction of the P value. In addition, there was no correlation between these antigens and clinical parameters. These results indicate that HLA class II alleles may not contribute to a strong susceptibility to melanoma in the Japanese
Molecular analysis of six dog leukocyte antigen class I sequences including three complete genes, two truncated genes and one full‐length processed gene
Authors: Burnett R. C. , DeRose S. A. , Wagner J. L , Store R.
Published: December 2008

We have isolated six distinct dog leukocyte antigen (DLA) class I sequences. An additional functional nonclassical class I gene, DLA‐79, was characterized previously. This brings the number of isolated canine class I sequences to seven. These seven loci account for nearly all class I sequences detected in genomic DNA by Southern blotting. With approximately seven members, the class I gene family in dog is considerably less complex than in either human or mouse. Three of the six sequences described in this article contain complete class I genes. These genes have the typical arrangement of exons and introns, and their predicted protein sequences have the features expected of expressed class I molecules. Two sequences contain truncated genes. These genes, having only partial class I homology and disruptive mutations, are clearly nonfunctional. The remaining locus contains a full‐length processed gene which is unique among characterized class I loci. Sequence comparisons were performed to examine evolutionary relationships among the family members. Two sequences, DLA‐64 (complete) and ‐53 (truncated), appear to be chimeras presumably formed by interlocus recombination. Using the reverse tran‐scription‐polymerase chain reaction, mRNA originating from each of the three complete genes was identified in canine peripheral blood leukocytes. DLA‐79 specific transcripts were identified previously. Thus, each of the four complete canine class I genes is transcribed in vivo.
HLA‐DQB1*0304‐DRB1*0408 haplotype associated with insulin–dependent diabetes mellitus in populations in the eastern Baltic region
Authors: Ilonen J. , Koskinen S. , Nejentsev S. , Sjöroos M. , Knip M. , Schwartz E. I. , Adojaan B. , Kovalchuk L. , Sochnevs A.
Published: December 2008

The rare HLA‐DQB1*0304 allele was found increased among IDDM patients in the populations of the eastern Baltic region. Its frequency among IDDM patients was 4.5% (20/443) compared to 1.1% (9/853) in healthy controls in the combined series of Estonian, Latvian and St. Petersburg Russian populations (P=0.0001). HLA‐DQB1*0304 in these populations was associated with DRB1*0408, and the haplotype was further characterized by a B35 allele and a typical combination of microsatellite markers from the TNF gene region. The result is compatible with the significance of the 57th amino acid in the DQ β‐chain but also emphasizes the importance of alleles in other HLA loci adjacent to DQ in the determination of IDDM susceptibility.
Molecular characterization of HLA‐C incompatibilities in HLA‐ABDR‐matched unrelated bone marrow donor‐recipient pairs
Authors: Grundschober C. , Rufer N. , Sanchez‐Mazas A. , Madrigal A. , Jeannet M. , Roosnek E. , Tiercy J.‐M.
Published: December 2008

While the influence of HLA‐AB and ‐DRB1 matching on the outcome of bone marrow transplantation (BMT) with unrelated donors is clear, the evaluation of HLA‐C has been hampered by its poor serological definition. Because the low resolution of standard HLA‐C typing could explain the significant number of positive cytotoxic T lymphocyte precursor frequency (CTLpf) tests found among HLA‐AB‐subtype, DRB1/B3/B5‐subtype matched patient/donor pairs, we have identified by sequencing the incompatibilities recognized by CD8+ CTL clones obtained from such positive CTLpf tests. In most cases the target molecules were HLA‐C antigens that had escaped detection by serology (e.g. Cw*1601, 1502 or 0702). Direct recognition of HLA‐C by a CTL clone was demonstrated by lysis of the HLA class I‐negative 721,221 cell line transfected with Cw*160l cDNA. Because of the functional importance of Cw polymorphism, a PCR‐SSO oligotyping procedure was set up allowing the resolution of 29 Cw alleles. Oligotyping of a panel of 382 individuals (including 101 patients and their 272 potential unrelated donors, 5 related donors and 4 platelet donors) allowed to determine HLA‐C and HLA A‐B‐Cw‐DRB1 allelic frequencies, as well as a number of A‐Cw, B‐Cw, and DRB1‐Cw associations. Two new HLA‐Cw alleles (Cw*02023 and Cw*0707) were identified by DNA sequencing of PCR‐amplified exon 2‐intron 2‐exon 3 amplicons. Furthermore, we determined the degree of HLA‐C compatibility in 287 matched pairs that could be formed from 73 patients and their 184 potential unrelated donors compatible for HLA‐AB by serology and for HLA‐DRB1/ B3/B5 by oligotyping. Cw mismatches were identified in 42.1% of these pairs, and AB‐subtype oligotyping showed that 30% of these Cw‐incompatible pairs were also mismatched for A or B‐locus subtype. The degree of HLA‐C incompatibility was strongly influenced by the linkage with B alleles and by the ABDR haplotypes. Cw alleles linked with B*4403, B*5101, B18, and B62 haplotypes were frequently mismatched. Apparently high resolution DNA typing for HLA‐AB does not result in full matching at locus C. Since HLA‐C polymorphism is recognized by alloreactive CTLs, such incompatibilities might be as relevant as AB‐subtype mismatches in clinical transplantation.
Induction of nuclear factor kB/Rel nuclear activity in human peripheral blood T lymphocytes by anti‐HLA class I monoclonal antibodies
Authors: Turco M. C. , Romano M. F. , Lamberti A. , Petrella A. , Bisogni R. , Sun S.‐C. , Ferrone S. , Bonelli P. , Cerra M. , Venuta S.
Published: December 2008

Monoclonal antibodies against either monomorphic or polymorphic determinants of class I antigen induced in PBMC and highly purified T lymphocytes the nuclear activity of NF‐kB/Rel complexes. These included both p50/p50 and p50/p65 dimers, recognized by specific antibodies in EMSA. The induced complexes were detectable in extracts of cells incubated with anti‐class I monoclonal antibody (mAb) for 1.5 h; the induction was maximal at 5 h, persistent at 16 h and no longer observed at 40 h. The mAb failed to induce NF‐kB/Rel nuclear activity in cells incubated in the presence of 3, 4‐dichloroisocoumarin, an inhibitor of IkB‐α degradation. Together, these results suggest that class I triggering can induce the activity of NF‐KB/Rel nuclear activity in peripheral blood T lymphocytes, thereby modulating the expression of genes regulated by these transcription factors.
Absolute linkage of celiac disease and dermatitis herpetiformis to HLA‐DQ
Authors: Balas A. , Vicario J. L. , Zambrano A. , Acuña D. , Garcfa‐Novo D.
Published: December 2008

This report shows the absolute genetic linkage of celiac disease (CD) to the HLA‐DQ region, and supports the fact that dermatitis herpetiformis (DH) follows the same pattern of HLA‐mediated susceptibility in extensive series of Caucasian Spanish patients. Ninety‐five percent of CD (201 of 212) and 100% of DH (55) patients could produce DQã1*0501‐DQβ1*02 heterodimers. Negative CD patients for this combination were mostly DR4‐DQ8 (DQA1*03‐DQB1*0302) (9 of 11), along with a restricted number of complementary chromosomes. Comparison of observed and expected DQA1‐DQB1 genotype distributions (Hardy‐Weinberg equilibrium) showed that the excess of patients with DQB1*02 in double doses would be the consequence for which this allele should be complemented by DQA1*0501. Homozygosity for DQA1*O501 would restrain susceptibility to CD and DH.
Heat shock protein 70 gene polymorphisms in rheumatoid arthritis
Authors: Vinasco J. , Beraun Y. , Nieto A. , Fralle A. , Pareja E. , Mataran L. , Martin J.
Published: December 2008

A role for heat shock proteins (hsp) in rheumatoid arthritis has been suggested. In addition, the specific binding of human HSP70 protein to QKRAA and RRRAA motifs within the HV3 region of disease‐associated DRB1*0401 and DRB1*1001 molecules, respectively, has been proposed as being relevant to rheumatoid arthritis. The purpose of this work was to analyze the influence of HSP70 gene polymorphism on the susceptibility to or severity of rheumatoid arthritis and to investigate the possible contribution of these HSP70 polymorphisms in determining HLA‐DRB1*0401/*1001 disease association. The frequencies of the HSP70‐1, HSP70‐2 and HSP70‐hom genotypes were analyzed by PCR‐RFLP using BsrBI, PstI and NcoI enzymes, respectively, in patients with heumatoid arthritis and in healthy controls. No significant differences were observed when HSP70 alleled istribution between the groups under study were compared. Moreover, we did not observe any significant difference in HSP70 allele frequencies between patients positive for HLA‐DRB1*0401/*1001 alleles and matched controls. Our data indicate that HSP70 gene polymorphisms do not appear to be relevant in the susceptibility to or severity of rheumatoid arthritis.
High resolution HLA‐C typing by PCR‐SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology
Authors: Bunce M. , Barnardo M. C. N. M. , Procter J. , Marsh S. G. E. , Vilches C. , Welsh K. I.
Published: December 2008

Recent evidence indicates that HLA‐C molecules are biologically relevant by eliciting T‐cell responses and exerting control over NK cell function. In addition, HLA‐C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA‐C has however proved difficult because detection of HLA‐C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA‐C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA‐C blank alleles. Recently, amplification of DNA using sequence‐specific primers (PCR‐SSP) has proved a reliable, accurate and rapid method for medium resolution HLA‐C typing. We have now developed high resolution HLA‐C typing by PCR‐SSP utilizing allele and group‐specific PCR‐SSP reactions which can identify all HLA‐C alleles (except non‐coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA‐C, PCR‐SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA‐C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High‐resolution HLA‐C typing of 112 International Histocompatibility Workshop cell lines has also been performed
Proteasome subunits, low‐molecular‐mass polypeptides 2 and 7 are hyperexpressed by target cells in autoimmune thyroid disease but not in insulin‐dependent diabetes mellitus: implications for autoimmunity
Authors: Vives‐PI M. , Vargas F. , James R. F. L. , Trowsdale J. , Costa M. , Sospedra M. , Somoza N. , Oblols G. , Tampé R. , Pujol‐Borrell R.
Published: December 2008

Autoimmune thyroid diseases (AITD) and insulin‐dependent diabetes mellitus (IDDM) are two autoimmune syndromes of unknown etiology with common immune features. One is that the target cells, thyrocytes and pancreatic islet beta cells respectively, hyperexpress several proteins encoded in the HLA region: HLA class I, HLA class II and transporter associated with antigen processing (TAP‐1); the clinical course and many aspects of the immunopathology are, however, quite different. Low‐molecular‐mass polypeptides 2 and 7 (LMP2 and LMP7) are proteasome subunits that increase the efficiency of endogenous antigen processing and are encoded in close vicinity to the TAP genes. We investigated whether LMP2 and LMP7 are hyperexpressed in thyrocytes and islet cells in AITD and IDDM. Thyroid tissue from Graves' disease patients (GD, n=8) and Hashimoto thyroiditis (HT, n=1) and pancreatic tissue from IDDM patients (n=4) as well as control tissues were examined by the two‐color indirect immunofluorescence technique. The results demonstrate that, in normal glands, thyrocytes and pancreatic islet cells express comparable moderate to low levels of LMP2 and LMP7. In AITD and IDDM, expression of LMP2/7 in the endocrine cells was disparate: while in AITD glands there was hyperexpression of LMP2 and 7 parallel to that of HLA class I and TAP‐1, in the islet cells of recent onset diabetic pancreases (n=2) the level of LMP2 and 7 expression was totally normal, including islets that were infiltrated by lymphocytes and hyperexpressed HLA class I and TAP‐1. These observations suggest different mechanisms of endogenous peptides generation at the target cells in AITD from IDDM. Since this is a key step for the maintenance of peripheral tolerance, it may help to understand some of the different clinical features of the two autoimmune diseases.
Episodic evolution and turnover of HLA‐B in the indigenous human populations of the Americas
Authors: Parham P. , Arnett K.L. , Adams E.J. , Little A.‐M. , Tees K. , Barber L. D. , Marsh S.G.E. , Ohta T. , Markow T. , Petzl‐Erler M. L.
Published: December 2008

Nucleotide sequences were determined for the HLA‐A, B and C alleles of three populations of Amerindians: the Havasupai tribe from North America, and the Guarani and Kaingang tribes from South America. All 15 Havasupai alleles are found in Eastern Hemisphere populations, whereas the Guarani and Kaingang each have six alleles that appear to be present only in the Western Hemisphere. Nine of the “new” alleles come from HLA‐B, one comes from HLA‐A and one from HLA‐C: ten appear to be the result of recombination and one the result of point substitution. Of the 14 Guarani alleles and 16 Kaingang alleles, only four are held in common. Despite their differences, the three tribes possess comparable numbers of HLA class I alleles, revealing a trend for “allele turnover”, in which new alleles tends to supplant older alleles rather than supplement them. Although many new HLA‐B alleles have been produced in Latin America, their net effect has been to differentiate populations, not to increase allele diversity within a population. From sequence comparisons, the Amerindian subset of HLA class I allotypes appears to cover the overall ranges of peptide binding specificity, natural killer‐cell interactions, and CD8 interactions, that are found in all HLA class I. The recombinations that produced the new alleles of the Kaingang and Guarani class I are predicted to have modulated these functional properties rather than radically change them. Exchange of Bw4 and Bw6 motifs by recombination are noticeably absent in the events forming new alleles in America, whereas they have been the most common of recombinations elsewhere.
Further diversification of the HLA‐B locus in Central American Amerindians: new B*39 and B*51 alleles in the Kuna of Panama
Authors: Iwanaga K. K. , Eberle M. , Kolman C. J. , Bermingham E. , Watkins D. I.
Published: December 2008

Several new HLA‐B locus alleles have been discovered in South American Amerindians. By contrast, analysis of the MHC class I alleles of North American native populations has revealed few new HLA‐B alleles. This suggests that the HLA‐B locus is evolving rapidly in South American populations. Here we describe the HLA‐B locus alleles present in individuals from a Central American tribe, the Kuna of Panama. Using a sequencebased typing technique that separates alleles by denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we determined the HLA‐B alleles from eight Kunas. Two of the HLA‐B alleles present in the Kuna have been previously described in other South American Amerindian populations; one allele has been characterized in a Mexican‐American. We characterized two new HLA‐B alleles in the Kuna, HLA‐B*3911 and HLA‐B*5110. HLA‐B*3911 differed from HLA‐B*3905 by only a single nucleotide substitution in exon 3. This substitution resulted in an amino acid replacement of leucine by arginine at residue 156 in the alpha 2 domain. Such a change may affect the repertoire of peptides that are bound by this molecule. HLA‐B*5110 differed significantly from other HLA‐B*51 alleles in that it is the result of an unusually large intra‐locus recombination event of minimally 216 nucleotides. This recombination results in an allele that is part HLA‐B*51 and part HLA‐B*40. Thus, more dramatic recombination events may also play a role in the rapid evolution of the HLA‐B locus in Amerindians.
Mapping epitopes to distinct regions of the extracellular domain of endoglin using bacterially expressed recombinant fragments
Authors: Pichuantes S. , Vera S. , Bourdeau A. , Pece N. , Kumar S. , Wayner E.A. , Letarte M.
Published: December 2008

Endoglin (CD 105) is a homodimeric cell surface component of the TGFß1 receptor complex, which is expressed at high levels on vascular endothelium and at lower levels on activated monocytes. It is also the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1. To date, each family has a distinct endoglin mutation, most of which generate premature stop codons. The purpose of the current study was to identify monoclonal antibodies capable of binding to normal and mutated forms of the protein. We generated stable transfectants of fulllength human endoglin in murine fibroblasts and engineered and expressed in bacteria several fragments of the extracellular domain. Relatively pure polypeptides were recovered with good yield from inclusion bodies and were tested by ELISA and Western blot; 11 monoclonal antibodies were shown to react specifically with the endoglin transfectants. Ten of these monoclonal antibodies reacted with the bacterial fragments, and their epitopes were assigned to 3 distinct regions of endoglin. Monoclonal antibodies P3D1, TEC4 and GRE reacted with the N‐terminal region of 204 amino acids encoded by exons 1 to 5. Monoclonal antibodies P4A4, 44G4, E‐9, MAEND3 and PN‐E2 all bound to a region of 54 amino acids encoded mostly by exon 7. Monoclonal antibodies CLE4 and RMAC8 reacted with the C‐terminal region of the extracellular domain, coded for by exons 8 to 12. Knowing the localization of these epitopes will facilitate the structural and functional analysis of normal and mutated forms of endoglin.
DNA typing of HLA class I (HLA‐A) and class II genes (HLA‐DR, ‐DQ and ‐DP) in Japanese patients with gastric cancer
Authors: Ohmori M. , Yasunaga S. , Maehara Y. , Sugimachi K. , Sasazuki T.
Published: December 2008

Gastric cancer is multifactorial disease and several reports have described genetic factors involved in pathogenesis of gastric cancer. Recently, it was reported that HLA class II gene DQB1*0301 was strongly associated with gastric cancer in Caucasian population (20). We performed DNA typing of HLA class I (HLA‐A) and HLA class II genes (HLA‐DR, DQ and DP) to elucidate the HLA alleles or HLA haplotypes associated with gastric cancer in Japanese population using polymerase chain reaction‐sequence‐specific oligonucleotide probe analysis in 88 unrelated patients with gastric cancer and 525 unrelated healthy controls. We observed slight difference in frequencies of some HLA alleles and haplotypes between gastric cancer patients and controls; however, after Bonferroni correction, statistical significance was not confirmed. It is possible that environmental factors such as diet cover the contribution of genetic factors to the disease in Japanese population, which has a higher frequency of gastric cancer than do Caucasian populations, most likely due to more exposure to environmental risk factors.
HLA class I DNA typing of 215 “HLA‐A, ‐B, ‐DR zero mismatched” kidney transplants
Authors: Mytilineos J. , Lempert M. , Middleton D. , Williams F. , Cullen C. , Scherer S. , Opelz G.
Published: December 2008

DNA typing for HLA class II improves the typing quality and this was shown previously to be relevant for kidney graft survival. In this project we addressed the question whether molecular typing for HLA class I also increases the efficacy of HLA matching in kidney transplantation. 215 HLA‐A,‐B,‐DR zero‐mismatched donor/recipient pairs as defined by serological typing were selected. Retrospective HLA‐A and HLA‐B typing was performed both by the PCR‐SSP and the PCR‐SSOP method. DNA typing for HLA‐A revealed discrepant results to serology in 5.7% of the donors and 2.8% of the recipients. HLA‐B typing discrepancies were found in 6.6% of the donors and 5.6% of the recipients. 10.4% of the donors and 6.5% of the recipients showed either an HLA‐A or an HLA‐B discrepancy. Nearly one‐third of the HLA‐A discrepancies affected A19 splits. The most common reason for HLA‐A discrepancies was the erroneous assignment of serological blanks, whereas HLA‐B errors were caused mainly by the assignment of incorrect specificities. DNA typing allowed the definition of HLA‐A and ‐B split specificities in all 118 “splitable” cases for which only broad specificities were reported based on serological typing. A total of 183 DNA class I compatible transplants had a 15% higher one‐year graft survival rate than 32 transplants for which DNA typing revealed a class I incompatibility.
A 25% error rate in serologic typing of HLA‐B homozygotes
Authors: Lorentzen D. F. , Iwanaga K. K. , Meuer K. J. , Moritz T. L. , Watkins D. I.
Published: December 2008

The microlymphocytotoxicity technique has been the accepted method for HLA class I typing since the early 1960s. However, it is often difficult to distinguish two related alleles expressed in an individual due to the cross‐reactive nature of the alloantibodies used in this technique. This is especially evident at the HLA‐B locus, whose more than 180 alleles fall into only 4 major interrelated cross‐reactive antigen groups. To estimate the error rate in serologic typing due to the cross‐reactive nature of sera, we used polymerasee chain reaction with sequence‐specific primers (PCR‐SSP) amplification to retype 40 individuals who were previously typed as serologic HLA‐B locus homozygotes. PCR‐SSP revealed that 10 of these 40 individuals (25%) were actually heterozygous at their HLA‐B locus The HLA‐B locus alleles of 9 of these 10 discrepant individuals were further analyzed by denaturing gradient gel electrophoresis followed by direct sequencing. The sequence analysis confirmed that all nine individuals were indeed HLA‐B locus heterozygotes. This surprisingly high error rate in serologic definition of HLA‐B molecules argues for the use of rapid DNA‐based techniques in HLA class I typing, even in the setting of solid organ transplantation.
HLA‐A typing: comparison between serology, the amplification refractory mutation system with polymerasee chain reaction and sequencing
Authors: Pera C. , Delflno L. , Morabito A. , Longo A. , Johnston‐Dow L. , White C. B. , Colonna M. , Ferrara G. B.
Published: December 2008

In this study we typed HLA‐A polymorphisms by a new sequence‐based typing (SBT) method, which involved one PCR reaction and four sequencing reactions covering exon 2 and exon 3. This method allowed complete identification of all known HLA‐A alleles and revealed the presence of a new allele, named HLA‐A*2608. We also introduced sequencing of exon 4 for some samples in order to discriminate the allelic pairs that are identical in exon 2 and 3, thus improving SBT resolution. Finally, we compared the results obtained by SBT with data obtained by serological typing and the amplification refractory mutation system (ARMS‐PCR). Together, our results suggest that the SBT here described provides an optimal HLA‐A typing technique that may be useful in selecting donor‐recipient pairs in bone marrow transplantation between unrelated individuals.
Comparison of typing results by serology and polymerase chain reaction with sequence‐specific primers for HLA‐Cw in 650 individuals
Authors: Mytilineos J. , Christ U. , Lempert M. , Opelz G.
Published: December 2008

HLA‐Cw typing by standard serological techniques is associated with a high frequency of blanks, and reliable typing reagents for several of the Cw specificities are scarce. We evaluated the PCR‐SSP technique for Cw typing in 370 kidney transplant patients and 280 healthy blood donors. Serological typing of all individuals was performed in our laboratory from 1995 to 1997 using commercially available tissue‐typing trays. Comparison between serological and PCR‐SSP typing revealed a discrepancy rate of 33.6% (n= 94) in blood donors and 32.4% (n=120) in kidney recipients. Incorrect antigen assignments occurred only rarely (3.6% of the blood donors and 3.2% of the kidney recipients). The vast majority of discrepancies were due to antigens that were not detected serologically. In 26 individuals no Cw antigen was detected by serological typing, whereas PCR‐SSP showed 1 allele in 13 and 2 alleles in the other 13 cases. Another 269 individuals were typed serologically with one blank (presumably homozygous). Of these, only 108 were confirmed to be homozygous, whereas an additional Cw allele was found in the remaining 161 cases using the SSP technique. Most of the “missed” specificities (86.5%) were those for which serological reagents were not available (HLA‐Cw*12‐*17). The most commonly “missed” specificity was HLA‐Cw*1203, which occurred in 13.9% of the healthy blood donors. These results indicate that serological HLA‐Cw typing is insufficient for examining the clinical importance of HLA‐Cw matching in transplantation. Future studies based on molecular typing should allow the proper investigation of HLA‐Cw matching in kidney and bone marrow transplantation.
Expression characteristics of the human CD97 antigen
Authors: Eichler W. , Hamann J. , Aust G.
Published: December 2008

Molecules whose expression is increased upon stimulation on leukocyte populations are of considerable interest because insights into their structure and function extend knowledge of the intracellular and intercellular events that accompany cellular activation. One such molecule is CD97, a cell surface antigen that is rapidly upregulated upon activation on lymphocytes. Increasing CD97 levels on peripheral blood lymphocytes (PBL) induced by different stimuli were found to be largely independent on de novo RNA and protein synthesis during the early stage of activation. Thus, inhibition of CD97 surface expression by cycloheximide was not noticeable or insignificant for 15 min to 4 h after stimulation. Furthermore, a fraction of intracellular CD97 decreased within 2 h suggesting redistribution of CD97 protein. Later, de novo protein synthesis apparently contributes to the induction of high CD97 surface density and inhibition by cycloheximide was more pronounced. Upregulation of CD97 on PBL involves protein kinase C‐, tyrosine protein kinase‐ and Ca2+‐dependent intracellular pathways. The effect on CD97 surface expression of the phorbol ester, phorbol 12‐myristate 13‐acetate (PMA), is different in PBL and Jurkat T cells. Whereas it stimulated after 22 h strong CD97 increase on PBL it suppressed CD97 expression on Jurkat T cells. CD97 expression, which is strong and constitutive on myelo‐monocytic cells, is shown to be not leukocyte‐restricted. Thus, CD97 transcripts were found in most of the investigated nonhematopoietic cell types and CD97 protein was detected on the cell surface at low amount. In a previous report, sequence data of a CD97 cDNA suggested a protein homologous to the secretin receptor superfamily and consisting of 722 amino acids with 8 potential glycosylation sites. According to this finding, glycoproteins displaying apparent molecular weights in the region of 70–85 kDa were detected in all investigated cell types. These molecules may represent differentially glycosylated and sialylated molecular forms of the same polypeptide. The findings support the notion that CD97 is broadly distributed and possesses a differentially regulated expression behavior on leukocytes and non‐hematopoietic cells.
Major histocompatibility complex class II alleles in Kazak and Han populations in the Silk Route of northwestern China
Authors: Mizuki N. , Ohno S. , Ando H. , Sato T. , Imanishi T. , Gojobori T. , Ishihara M. , Ota M. , Geng Z. , Geng L. , Li G. , Kimura M. , Inoko H.
Published: December 2008

Genetic polymorphism of the HLA class II loci including the DRB1, DQA1, DQB1 and DPB1 genes was investigated by the polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) method in a Kazak population inhabiting the most northwestern part of China, Ürümqi in the Xinjiang Uygur Zizhiqu as well as in a Han population in the same area. Forty‐two Kazak and 59 Han unrelated volunteers were enrolled in this study. Among 51 DRB1 alleles tested, 29 alleles were detected, and DRB1*0301 (13.1%) and DRB1*07 (10.7%) in Kazak and DRB1*0901 (11.9%), DRB1*1501 (11.0%) and DRB1*07 (11.0%) in northwestern Han were highly predominant. In 8 DQA1 alleles detected, DQA1*0501 (29.8%) and DQAP0301 (23.8%) in Kazak, and DQA1*0301 (28.8%) and DQA1*0102 (19.5%) in northwestern Han were the most and the second most common alleles, respectively. Of 18 DQB1 alleles tested, 14 were observed, among which DQB1*0201 and DQB1*0301 were very frequent both in Kazak (23.8% and 21.4%, respectively) and northwestern Han (18.6% and 16.9%, respectively) populations. Of 37 DPB1 alleles tested, 14 were detected. Among them, the frequencies of DPB1*0401 (21.4%), DPB1*0501 (20.2%), DPB1*0402 (19.0%) and DPB1*0201 (16.7%) in Kazak, and those of DPB1*0501 (38.1%) and DPB1*0201 (16.1%) in northwestern Han were highly increased. Several three‐locus haplotypes were recognized to predominate significantly, namely DRB1*0301‐DQA1*0501‐DQB1*0201 (13.1%) and DRB1*0701‐DQA1*0201‐DQB1*0201 (8.3%) in Kazak; and DRB1*0901‐DQA1*0301‐DQB1*0303 (11.9%) and DRB1*0701‐DQA1*0201‐DQB1*0201 (10.2%) in northwestern Han. The dendrogram constructed by the neighbor‐joining (NJ) method based on the allele frequencies of the DRB1, DQA1, DQB1 and DPB1 genes of 12 representative populations all over the world including northern Han, southern Han, Manchu and Japanese suggested that Kazak and northwestern Han were the closest to each other, but Kazak was a little farther from the Asian ethnic groups than northwestern Han.
Complete HLA‐A DNA typing using the PCR‐RFLP method combined with allele group‐ and sequence‐specific amplification
Authors: Moribe T. , Kaneshige T. , Inoko H.
Published: December 2008

We have established a practical method of complete high‐resolution typing for all HLA‐A alleles using the polymerase chain reaction (PCR)‐restriction fragment‐length polymorphism (RFLP) technique combined with allele group‐ and sequence‐specific amplification. The second and third exons of the HLA‐A gene, in which most allelic variations are observed, were separately amplified by PCRs with 3 and 4 group‐specific primer pairs, respectively. Each PCR‐amplified product was digested by allele‐specific restriction endonucleases and then subjected to electrophoresis on a 10% polyacrylamide gel. In this way, 62 out of 79 HLA‐A alleles could be discriminated by the RFLP patterns derived from the genetic polymorphism in the exon 2 and 3 domains. The remaining 17 alleles could be defined unequivocally by either PCR‐RFLP analysis after exon 4 amplification or PCR analysis with sequence‐specific primers (SSP). By this method, complete HLA‐A genotyping for all homozygous and heterozygous combinations can be accomplished, establishing technically simple, economical and practical routine typing of the HLA‐A gene, especially for small samples.
Characterization of rabbit antisera elicited with human LMP2‐ and LMP7‐specific peptides and recombinant proteins
Authors: Dellaratta D.V. , HIcklin D.J. , Kishore R. , Kageshita T. , Ferrone S.
Published: December 2008

Anti‐human LMP2 and anti‐human LMP7 sera with a titer of at least 1:10,000 were developed by immunizing rabbits with LMP2‐ and LMP7‐specific peptides corresponding to C‐terminal regions of each subunit or with TrxLMP2 and TrxLMP7 recombinant proteins. IgG antibodies elicited by immunization with LMP‐specific peptides or recombinant proteins displayed reactivity with their respective immunogens in ELISA. Furthermore, antibodies elicited with both types of immunogens recognize native and recombinant LMP2 and LMP7 subunits in Western blotting and are able to immunoprecipitate LMP2 and LMP7 as components of the 20S proteasome from lymphoid cell lysates. In ELISA, a subpopulation of the antibodies generated with LMP peptides and recombinant proteins corresponding to one LMP subunit is cross‐reactive with the other one. This antibody subpopulation was not detectable in the affinity‐purified antibody populations isolated by passing antisera over the corresponding immunogen. Neither anti‐LMP2 nor anti‐LMP7 sera displayed cross‐reactivity with the homologous proteasome subunits Delta and MB1. In immunohistochemical reactions affinity‐purified anti‐LMP2 and anti‐LMP7 antibodies stained cells in both frozen and formalin‐fixed tissue sections of normal skin. These results indicate that the anti‐LMP2 and anti‐LMP7 sera elicited with peptides and recombinant proteins are both useful reagents for biochemical characterization of LMP2 and LMP7 and to analyze their expression in normal and transformed cells.
HLA class I and class II allele distribution in the Bubi population from the island of Bioko (Equatorial Guinea)
Authors: de Pablo R. , García‐Pacheco J.M. , Vilches C. , Moreno M.E. , Sanz L. , Rementería M.C. , Puente S. , Kreisler M.
Published: December 2008

We determined the HLA frequency distribution in a sample of 100 Bubi individuals born on the island of Bioko (Equatorial Guinea). HLA‐A, ‐B and ‐C typing was performed by serology and PCR‐SSR DRB1/3/4/5, DQB1 and DQA1 alleles were determined by PCR‐SSOP. The HLA allele distribution of this population group resembles those found in other Bantuspeaking groups; however, the higher frequency of A30, A32, B44, DRB1*1301 in the Bubi with respect to other Bantu groups and the absence of DR4 deserve special mention. The cloning and sequencing of class I and II genes in this population allowed the description of five new allelic variants: B*4407, Cw*0706, Cw*1801, Cw*1802 and DQB1*0612 and five confirmatory sequences: B*3910, B*5703, B*8101, Cw*1203 and Cw*1701. The following new HLA‐C,B haplotypes have been found in Bubi: Cw*08B*57, Cw*18‐B*57, Cw*0302‐B*53, Cw*07‐B*53 and Cw*1601‐B*63. The most frequent seven‐locus haplotype is: A*30‐Cw*17‐B*42‐DRB1*1102DRB3*0202‐DQA1*05‐DQB1*0301. In terms of genetic distance, the Bubi are closer to other Bantu groups than to West African populations.
Genetic polymorphism in intron 6 of the LMP7 gene in Japanese and its association with sarcoidosis
Authors: Ishihara M. , Ohno S. , Ishida T. , Naruse T. , Inoko H.
Published: December 2008

Genetic polymorphism in intron 6 of the LMP7 gene was investigated using polymerase chain reaction‐restriction fragment length polymorphism in 90 unrelated healthy Japanese controls and 66 Japanese patients with sarcoidosis. Four alleles, including two new ones recently identified in Koreans, LMP7*C and LMP7*D, were found in the Japanese population. The frequency of LMP7*C in the sarcoidosis patients was higher than in the healthy controls. However, this difference might be explained by a secondary association with HLA‐DRB1*08 in the HLA‐DRB1 gene, which is thought to be the gene primarily responsible for susceptibility to sarcoidosis.
High‐resolution HLA‐DQB1 typing using the polymerase chain reaction and sequence‐specific primers
Authors: Mullighan C.G. , Bunce M. , Welsh K.I.
Published: December 2008

Polymorphism at HLA‐DQB1 is known to influence tissue compatibility and disease susceptibility; however, current DQB1 typing methods are unable to distinguish the 32 currently recognized DQB1 alleles. We have developed a 32‐reaction PCR‐SSP method capable of differentiating all DQB1 alleles that differ in amino acid sequence. This method can resolve all heterozygous combinations of DQB1 alleles, with the exception of several combinations involving alleles not thus far detected in Caucasoid populations.
Characterization and distribution of HLA‐B*5002 in a Spanish population sample
Authors: Balas A. , Santos S. , García‐Sánchez F. , Lillo R. , Merino J.L. , Vicario J.L.
Published: December 2008

Abstract: HLA‐J345, in contrast to B44, does not show molecular polymorphism. We have found a group of Caucasian Spanish individuals, sero‐logically typed as B45, showing an unexpected HLA‐B12 PCR‐SSO subtyp‐ing pattern. Complete coding region sequencing and B45 subtyping by PCR‐SSO demonstrated that the j345 serologic specificity is constituted by two molecular alleles: B*4501 and B*5002. B*5002 is recognized by polyclonal and monoclonal allosera against B12 and B45, whereas it is not detected by B21, B49, or B50 reagents, providing a new example of poor correlation between serology and structure. B*5002 explains an important subset (18%) of the B45‐positive individuals of the Spanish population studied, and almost half are included in a very infrequent haplotypic association, Cw6‐B*5002‐DRB1*0406‐DQA1*03‐DQB1*0402.