Interacting within metaphors

Serological analysis suggests the existence of a novel HLA-B39 subtype (HLA-B39N) in the Japanese population. To identify this novel allele, a gene encoding HLA-B39N was cloned and the exons were sequenced. A gene encoding HLA-B39N (B*3904) and B*39011 differs by two nucleotide substitutions at codons 11 and 12 whereas B*3904 and B*39013 differ by three nucleotide substitutions at codons 11, 12, and 312. One nucleotide difference at codon 11 produces a change from serine in B*3901 to alanine in B*3904 whereas another difference at codon 12 changes valine in B*3901 to methionine in B*3904. The residues 11 and 12 are located on the beta-sheet out of the peptide-binding floor and are completely buried in the molecule. These results suggest that the substitutions at these residues alter the conformation of other residues forming epitopes of alloantibodies. Analysis of HLA-B*3901 genes in the Japanese population showed that both B*39011 and B*39013 were observed in the Japanese population. The present study suggests that B*3904 may have evolved from B*39011 rather than B*39013.     

Repair of complete atrioventricular septal defect with severe pulmonary hypertension--effect of re-pulmonary artery banding and analysis of lung biopsy: a case report

HLA-B18 is a well defined Bw6-associated serologic specificity. Up to now, four different sequences have been characterised in Caucasian populations (B*1801,3,4,5), and one in Orientals (B*1802). We report a new HLA-B18 subtype (B*1806) which was serologically detected in a Spanish Caucasian individual as a B18 Bw4-associated antigen. Complete coding region sequencing showed that B*1806 differs from B*1801 in a unique nucleotide at position 299 (A to T), giving rise to an amino acid replacement in residue 76 (glutamic acid to valine) placed at the alpha1 domain. Therefore, in contrast to the serologic results, B*1806 possesses the canonical Bw6 motif at position 77-83. Subsequent flow cytometric assays proved that B*1806 evidences neither Bw4 nor Bw6 epitopes. Only three additional HLA-B alleles encode valine at codon 76, B*4601, B*7301 and B*5503, and like B*1806, all of them would include a Bw6 motif associated to the negative recognition by Bw6 antibodies. These findings support that valine at position 76 will modify the Bw6 epitope drastically, and suggest that this group of HLA-B alleles would define a third, Bw4 and Bw6-negative, lineage of molecules. Furthermore, valine 76 will also prevent the binding of Bw6 antibodies to those HLA-C antigens with the canonical Bw6 epitope (Cw*1,3,7,8,12,13,14,16).     

Binding of the pili of Pseudomonas aeruginosa to a low-molecular-weight mucin and neutral cystatin of human submandibular-sublingual saliva

The HLA-B*27 group of alleles has been extensively studied due to the association of particular B*27 alleles with ankylosing spondylitis (AS). We describe here an HLA-B*27 allele (B*2712) encoding an antigen that lacks reactivity with B27 monoclonal antibodies (moabs) and alloantisera but reacts with some B40/B60 moabs and alloantisera and expresses the Bw6 public epitope. This allele was discovered by the segregation of an HLA-B allele undetectable by PCR-SSP within a Caucasian family from the British population referred for routine bone marrow transplant HLA typing and found in the haplotype A*29; B*2712; Cw*1203; DRB1*13; DQB1*0603. Serological typing showed a lack of reactivity with four B27 moabs and four alloantisera but positive reactivity with moabs and alloantisera specific for B40/B60 and Bw6 public epitopes. Subsequent sequencing showed the closest homology was with B*2708 with three mismatches in exon 2 at positions 204, 209 and 210. The intron 2 sequence was identical with other B*27 lineage alleles including a 2 base pair deletion at positions 95 and 96. The relationship between HLA-B*2712 and reported B60 associations with susceptibility to AS remains to be determined.     

What is the diagnosis? A 2-year-old child refuses to walk

A new DPB1 allele has been identified in a Caucasoid individual, DPB1*7601. The sequence of the complete second exon has been confirmed by cloning and subsequent sequencing. This allele differs by one amino acid, at codon 36, from DPB1*1401, as indicated by SBT and PCR-SSP analysis. The amino-acid motif introduced by the change is shared by DPB1*0401 and some rare alleles. It remains unclear whether the change is due to interallelic microgen conversion or a single point mutation.     

Less common causes of carpal tunnel syndrome

A new DRB3*02 allele (DRB3*0207) was detected in a female Luxembourg Caucasian blood donor by sequence-based typing. The new allele differs from DRB3*0202 by two substitutions in codon 57 resulting in an amino acid change from a charged aspartic acid to a neutral valine. This is the first example of a DRB3 allele pair differing only at codon 57.     

Sequence-based typing provides a new look at HLA-C diversity

Although extensive HLA-A and HLA-B polymorphism is evident, the true diversity of HLA-C has remained hidden due to poor resolution of HLA-C Ags. To better understand the polymorphic nature of HLA-C molecules, 1823 samples from the National Marrow Donor Program research repository in North America have been typed by DNA sequencing and interpreted in terms of HLA-C diversification. Results show that HLA-Cw*0701 was the most common allele with a frequency of 16%, whereas 28% of the alleles typed as Cw12-18 (serologic blanks). The frequency of homozygotes was 9.8% as compared with previous studies of 18% for sequence-specific primers and 50% for serology. Most startling was the frequency at which new alleles were detected; 19 new HLA-C alleles were detected, representing a rate of approximately 1 in 100 samples typed. These new HLA-C alleles result from 29 nucleotide substitutions of which 4 are silent, such that coding substitutions concentrated about the Ag-binding groove predominate. Polymorphism at the HLA-C locus therefore resembles that at the HLA-A and HLA-B loci more than previously believed, indicating that antigenic stress is driving HLA-C evolution. However, sequence conservation in the alpha-helix of the first domain and a clustering of unique amino acids around the B pocket indicate that HLA-C alleles respond to antigenic pressures differently than HLA-A and HLA-B. Finally, because the samples characterized were predominantly from Caucasians, we hypothesize that HLA-C polymorphism will equal or exceed that of the HLA-A and -B loci as DNA sequence-based typing is extended to include more non-Caucasian individuals.     

Influence of coding region polymorphism on the peripheral expression of a human TCR V beta gene

A number of human TCR V beta gene segments are reported to be polymorphic, with alleles differing by one or a small number of amino acid substitutions. In the absence of detailed structural information regarding the interaction of specific positions in the TCR with Ag or MHC, the significance of such variation is difficult to assess. In this report the relative use of the two common alleles of the human V beta 6.7 gene, 6.7a and 6.7b, which differ by two non-conservative amino acid substitutions, and the use of two common alleles of the V beta 12.2 gene, which differ by only silent substitutions, were measured in PBL derived from individuals heterozygous for these alleles. Equal use of V beta 12.2 alleles was observed, consistent with the inability of selection mechanisms to discriminate between the products of these alleles that are indistinguishable at the amino acid level. However, statistically significant skewing in the use of V beta 6.7 alleles was observed in 15 of 16 individuals studied. Expression levels for each allele ranged from 16 to 84% of the total V beta 6.7 signal in heterozygous individuals, with either the 6.7a or the 6.7b allele predominant in different individuals. Based on segregation studies in families, it seems unlikely that other unidentified polymorphism in the TCR beta locus, such as in the V beta 6.7 promoter, was responsible for the differential allele expression. Family studies provided no evidence for an association between specific HLA haplotypes and V beta 6.7 allele use. These results indicate that even modest allelic variation in human TCR V beta coding regions can have a significant impact on the expression of human V beta genes in the peripheral repertoire.     

Characterization of an HLA-B62 variant (B*1538) exhibiting an additional B52 serologic reactivity

Antigens bearing the B62 serologic specificity are a heterogeneous group being encoded by at least 10 alleles and are widespread in most populations including the Korean population (10.5%). This study characterized a new allele encoding a B62 molecule with extra B52 serologic reactivity from a Korean family and unrelated individuals. Based on the DNA sequence, it appears that the single nucleotide substitution at codon 171 (TAC-->CAC), resulting in an amino acid change from tyrosine to histidine, is responsible for creating the extra reactivity. B*1538 was confirmed by PCR-SSP using a primer annealing to codon 171 in two additional unrelated individuals also exhibiting the same serologic reaction pattern. The haplotype associated with the novel allele, A31-B*1538-Bw6-Cw3-DRB1*1101-DRB3*02-DQB1*0301, was identified in the family members and two unrelated individuals. The novel B*1538 allele and its associated haplotype adds to the HLA diversity in this population.     

Genomic analysis of the F subtypes of human complement factor B

Factor B of human complement is encoded within the Major Histocompatibility Complex (MHC) and is polymorphic, with up to 30 alleles defined by electrophoretic mobility. One of the most common alleles, BF*F, is subdivided into the FA and FB subtypes, which differ at the gene level by non-synonymous base substitutions in the seventh codon. We have found at this position a new restriction site polymorphism, as a Bsl I site absent from the FB allele. Using this restriction polymorphism, we have developed a method for BF F subtype determination, based on amplification by polymerase chain reaction of the 5' end of the BF gene, and digestion with Bsl I. This new method has been applied to a panel of 29 selected BF F individuals. A single strand DNA conformation analysis of the same region of the gene allowed us to confirm the above DNA-based BF F subtyping. During this study, two BF*F1 alleles showed discrepancies between protein and DNA typing, which were confirmed by our sequencing data. These were identical, in the 5' region, to BF*S and BF*FB genes, respectively. In a comparison with two protein subtyping methods, identical results were found for only one third of the selected samples. The conflicting results may arise, in part, from previously undescribed molecular heterogeneity within BF F subtypes, or from the presence of a null allele. Our new method allows BF*F subtyping to be used with confidence in the definition of disease-associated MHC haplotypes.     

Tobacco farmers and diversification: opportunities and barriers

PCR/SSOP typing methods were used to analyze the HLA Class II DRB1, DQA1, DQB1 and DPB1 loci of samples from three African American populations of Colombia. Forty samples from the Cauca (Pacific), and twenty samples each from the Choco (North Pacific Coast) and the Providencia (Caribbean island) populations, were collected and the Class II loci analyzed under the auspices of the Expedicion Humana. Despite the limited number of samples analyzed, the African Colombian populations exhibit a very high degree of class II polymorphism. A great diversity of DRB1 alleles was found, with representatives from all serological classes, including 19 DRB1 alleles in the Providencia, 16 in the Cauca and 14 in the Choco groups. In addition, a novel DQB1*02 allele (*0203) was found in two individuals from the Cauca population of the Pacific Coast. The sequence of the DQB1*0203 allele, associated with DR3, differs from DQB1*0201 by only one nucleotide substitution (C-->A) in the second position of codon 57, resulting in an Ala to Asp change. The addition of DQB1*0203 brings the total number of DQB1 alleles identified to date to 26. HLA class II diversity is much greater in these African Colombian populations than that seen in nearby Amerindian populations. Analysis of regional Colombian African American HLA population genetics is discussed with respect to the Colombian Amerindian HLA genetics described in an accompanying paper.     

Nutritional and metabolic support strategies

We report here the identification of four novel DRB alleles using a reverse hybridization (CANTYPE) assay. Molecular cloning and sequencing confirmed the initial unusual hybridization patterns. All four new alleles were detected during routine HLA typing for the Canadian Unrelated Bone Marrow Donor Registry. DRB1*0703 is identical to DRB1*0701 except for a single nucleotide substitution (AGA-->AGT), changing codon 29 from Arg to Ser, a so far undetected DRB polymorphism. DRB1*0817 differs from DRB1*0801 by a single nucleotide substitution (TAC-->TTC), changing codon 47 from Tyr to Phe. This polymorphism has not, until now, been identified in DRB1*08 alleles. Compared with DRB3*0301, DRB3*0302 contains a single nucleotide substitution (TAC-->CAC) at codon 30, changing the encoded Tyr to His. This polymorphism is typical for DRB3*02 alleles. DRB3*01014 is identical to DRB3*0101 except for a single silent nucleotide substitution (GGG-->GGA) at codon 84. This polymorphism has previously only been described for the DRB1*15012 allele. DRB1*0817, DRB3*0302 and DRB3*01014 may have arisen from gene conversion, but DRB1*0703 most likely was generated by a point mutation event. The DRB3*0302 allele was detected in two unrelated subjects, while the other three have each only been detected once.     

HLA-DRB1*04 and DRB1*14 alleles are associated with susceptibility to pemphigus among Japanese

It has previously been demonstrated that susceptibility to pemphigus vulgaris is associated with human leukocyte antigen (HLA)-DR4 serologic specificity among Ashkenase Jews, and with DR4 as well as DR6 (DR14) in other ethnic groups. We genotyped HLA-DRB1, DQA1, DQB1, and DPB1 alleles in 16 patients with pemphigus by polymerase chain reaction-restriction fragment length polymorphism, to find evidence of potential HLA class II allele associations with pemphigus in Japanese patients who have a relatively homogeneous ethnic background. All nine patients with pemphigus vulgaris and five of seven patients with pemphigus foliaceus carried one or two alleles of HLA-DRB1*04 (*0403, *0406) and HLA-DRB1*14 (*1401, *1405, *1406) subtypes. Sequence analysis of these DRB1*04 and DRB1*14 alleles revealed the amino acid homology of phenylalanine at position 26 and valine at position 86 with the DRB1*0402 allele that reportedly confers a strong susceptibility to pemphigus vulgaris in Ashkenazi Jews. Thus our findings, together with previous HLA studies on pemphigus vulgaris patients of different ethnic groups, suggest that HLA-DRB1*04 and DRB1*14 alleles are commonly associated with pemphigus vulgaris across racial barriers. These HLA-DRB1 alleles are likely to be also associated with pemphigus foliaceus. Further studies on more diverse ethnic populations will be helpful in determining the significance of the association between certain amino acid residues of the class II molecules and disease susceptibility to pemphigus vulgaris as well as pemphigus foliaceus.     

Molecular genetic analysis of the ABO blood group system: 4. Another type of O allele

We have encountered an allele which seems to be another type of O allele at the human histo-blood group ABO locus. We have determined the nucleotide sequence of this allele over the coding region in the last two coding exons. This allele does not possess the single-nucleotide deletion found common among all the O alleles previously analyzed. Compared with A1 allele, this allele has three nucleotide substitutions resulting in two amino acid substitutions. The introduction of these amino acid substitutions into the A1 transferase expression construct apparently abolished the enzymatic activity of A1 transferase.     

Contribution of DRB1*04 variants to predisposition to or protection from insulin dependent diabetes mellitus is independent of dq

Certain DQ alpha/beta dimeric molecules have been shown to play a major role in determining susceptibility or resistance to IDDM. Whether or not predisposition associated with DR4 haplotypes is exclusively due to linkage to DQB1*0302 and DQA1*0301 alleles is still a controversial issue. A modifying effect of certain DRB1*04 subtypes on the susceptibility encoded by DQ alleles is possible, since not all DRB1*04-DQB1*0302 haplotypes are associated with the disease. The distribution of DRB1*04 subtypes was analysed in 240 DR4-positive Caucasian IDDM patients and 110 DR4-positive healthy controls using allele-specific hybridization after genomic amplification. Although an important contribution to IDDM predisposition was encoded by the DQB1*0302 allele which was found in the majority of patients (94.2% vs 64.7% in controls, Odd's ratio OR = 8.8, P < 0.0001), differences between DRB1*04 variants persisted after the effect of the DQB1 locus was removed by matching patients and controls for DQB1*0302. Thus, the DRB1*0402 allele conferred a strong IDDM-predisposing effect (OR = 3.1, P < 0.02) which was highly significant in the absence of DR3 on the second haplotype (OR = 5.6, P < 0.0001) but was not visible among DR3/4 heterozygote individuals. Conversely, the DRB1*0404 allele conferred a strong protective effect (OR = 0.26, P < 0.0001) which was dominant even in the presence of the associated high risk DR3 haplotype (OR = 0.21, P < 0.03). These data indicate that DQ molecules are not the sole contributors to the DR4-associated IDDM predisposition, and that peculiar DR4 subtypes play a significant role in susceptibility to or protection from the disease. DRB1*0402 differs from DRB1*0404 by only two acidic residues at positions 70 and 71 within the peptide binding groove, instead of amide and basic amino acids. This might induce changes of peptide binding specificity that correlate with the genetic linkage of IDDM predisposition.     

HLA-B44-directed cytotoxic T cells associated with acute graft-versus-host disease following unrelated bone marrow transplantation

We describe the recipient of a marrow graft from an HLA-serologically identical unrelated donor from whom highly potent host-reactive CTL of donor origin were isolated in association with acute GVHD. Extensive sequence and biochemical analysis of the HLA complex of this donor and recipient revealed several disparities in class I and class II HLA with the potential to be recognized by T cells from the donor or the host. The donor-derived CTL exclusively recognized a class I HLA difference associated with HLA-B44. Nucleotide sequencing of donor and recipient cells revealed that the patient possessed the HLA-B*4402 allele recognized by IEF as B44.2 while the donor possessed HLA-B*4403 (IEF variant B44.1). These alleles differ at one amino acid residue located at position 156 in the alpha 2 domain. The donor-derived CTL were shown to be specific for B44.2 by blocking studies and by the lysis of five different B44.2+ unrelated cell lines, two of which were confirmed by sequencing to be homozygous for B*4402. A host-specific difference involving a HLA-DRB1 allele was not recognized by the CTL, neither did HLA differences unique to the donor HLA-B*4403 and HLA-DQ8 elicit a host response. These data show that certain HLA disparities may be tolerated at the same time that other disparities elicit a potent immunologic response. The chemical nature of the difference, its structural impact, as well as the conditions of transplant appear to influence the type of response which occurs.     

CYP2D6 phenotype-genotype relationships in African-Americans and Caucasians in Los Angeles

CYP2D6 genotyping (CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*13, CYP2D6*16 alleles and gene duplications) was previously performed on 1053 Caucasian and African-American lung cancer cases and control individuals and no significant difference in allele frequencies between cases and control individuals detected. We have carried out additional genotyping (CYP2D6*6, CYP2D6*7, CYP2D6*8, CYP2D6*9, CYP2D6*10, CYP2D6*17 alleles) and debrisoquine phenotyping on subgroups from this study to assess phenotype-genotype relationships. African-Americans showed significant differences from Caucasians with respect to frequency of defective CYP2D6 alleles, particularly CYP2D6*4 and CYP2D6*5. The CYP2D6*17 allele occurred at a frequency of 0.26 among 87 African-Americans and appeared to explain higher average metabolic ratios among African-Americans compared with Caucasians. CYP2D6*6, CYP2D6*8, CYP2D6*9 and CYP2D6*10 were rare in both ethnic groups but explained approximately 40% of higher than expected metabolic ratios among extensive metabolizers. Among individuals phenotyped with debrisoquine, 32 out of 359 were in the poor metabolizer range with 24 of these (75%) also showing two defective CYP2D6 alleles. Additional single strand conformational polymorphism analysis screening of samples showing large phenotype-genotype discrepancies resulted in the detection of three novel polymorphisms. If subjects taking potentially interfering drugs were excluded, this additional screening enabled the positive identification of 88% of phenotypic poor metabolizers by genotyping. This sensitivity was comparable with that of phenotyping, which identified 90% of those with two defective alleles as poor metabolizers.     

Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of .005 (*1x2), .013 (*2x2), and .001 (*4x2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T1957C), *2B (additional C2558T), and *4E (additional C2938T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EM/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment.     

Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities

A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.