A phylogenetic analysis of cattle DRB3 alleles with a deletion of codon 65

 One of the most common cattle major histocompatibility complex DRB3 alleles, ^* 0201, includes a deletion of codon 65 encoding one residue in the α-helical chain. The mutation is functionally interesting and is likely to influence peptide binding. Exon 2 of two additional del65 alleles, ^* 3301 and ^* 4101, have now been sequenced with the aim to investigate the evolutionary relationship of this allelic group. Despite a fairly large genetic distance between the three alleles (11–17 nucleotide substitutions causing 8–11 amino acid substitutions) we found clear indications of a common ancestry. The α-helical region was very similar or identical among the alleles whereas the β-strand region was quite divergent. The results indicated that interallelic recombination has contributed to the diversification of the del65 group. Deletion of codon 65 has also been found in a roe deer DRB1 allele and a cattle DQB3 allele. Sequence comparisons of the cattle and roe deer DRB del65 alleles refuted the possibility of a trans-species persistence of a del65 allelic lineage but the two species may share a short ancestral sequence motif including del65. In addition to del65, the cattle DQB3 allele did not show any striking sequence similarities to the DRB alleles. Received: 20 March 1997 / Revised: 17 June 1997     

Cw * 1701 defines a divergent African HLA-C allelic lineage

 The complete sequence of a new HLA-C allele, Cw ^* 1701, was determined from a South African Zulu individual. Unique features that distinguish Cw ^* 1701 from other HLA-C alleles include multiple point substitutions and an 18 nucleotide insertion in exon 5, which encodes the transmembrane domain. In a phylogenetic analysis of HLA-C sequences, Cw ^* 1701 forms a third, distinct allelic lineage. A comparison of the transmembrane domain of Cw ^* 1701 with other HLA-B and -C alleles reveals that duplications and deletions have been common in the evolution of these loci. A polymerase chain reaction based typing method was used to determine the distribution of this unusual allele in human populations. In contrast to the other two lineages of HLA-C alleles, the Cw ^* 17 lineage is found at high frequencies only in populations of African descent. In addition, the HLA-B/Cw ^* 17 haplotype diversity is higher in Africa. Received: 29 June 1996  / Revised: 20 December 1996     

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

 HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B ^* 3906 and B ^* 1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B ^* 5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B ^* 5101 and B ^* 52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B ^* 39061 and B ^* 39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B ^* 1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies. Received: 11 March 1997 / Revised: 29 May 1997     

Molecular, genetic and biochemical characterization of lactate dehydrogenase-A enzyme activity mutations in Mus musculus

Four independent heterozygous lactate dehydrogenase (LDH) mutations with approximately 60% of wild-type enzyme activity in whole blood have been recovered. The mutant line Ldh1 ^a2Neu proved to be homozygous lethal, whereas for the three lines Ldh1 ^a7Neu, Ldh1 ^a11Neu, and Ldh1 ^a12Neu homozygous mutants with about 20% residual activity occurred in the progeny of heterozygous inter se matings. However, the number of homozygous mutants was less than expected, suggesting an increased lethality of these animals. Various physicochemical and kinetic properties of LDH are altered. Exons of the Ldh1 gene were PCR amplified and sequenced to determine the molecular lesion in the mutant alleles. Ldh1 ^a2Neu carried an A/T → G/C transition in codon 112 (in exon 3), resulting in an Asn → Asp substitution; Asn112 is part of the helix αD, which is involved in the coenzyme-binding domain. Ldh1 ^a7Neu contained an A/T → C/G transversion within the codon for residue 194 in exon 4, causing an Asp → Ala substitution, which may affect the arrangement of the substrate-binding site. Three base substituions were discovered for the mutation Ldh1 ^a11Neu in exon 7: the transition C/G → T/A, a silent mutation, and two transversions C/G → A/T and C/G → G/C, both missense mutations, which led to the amino acid replacements Ala319 → Glu and Thr321 → Ser, respectively, located in the αH helix structure of the COOH tail of LDHA. We suggest that the mutation is the result of a gene conversion event between Ldh1 ^a wild-type gene and the pseudogene Ldh1-ps. The alteration Ile → Thr of codon 241 in exon 6 caused by the base pair change T/A → C/G was identified in the mutation Ldh1 ^a12Neu; IIe241 is included in the helix α2G, a structure that is indirectly involved in coenzyme binding. Each of the sequence alterations has a potential impact on the structure of the LDHA protein, which is consistent with the decreased LDH activity and biochemical and physiological alterations. Received: 3 July 1997 / Accepted: 30 September 1997     

Diversity associated with the second expressed H L A - D R B locus in the human population

Although diversity within the HLA-DRB region is predominantly focused in the DRB1 gene, the second expressed DRB loci, DRB3, DRB4, and DRB5, also exhibit variation. Within DRB1 ^* 15 or DRB1 ^* 16 haplotypes, four new variants were identified: 1) two new DRB5 alleles, DRB5 ^* 0104 and DRB5 ^* 0204, 2) a haplotype carrying a DRB1 ^* 15 or ^* 16 allele without the usual accompanying DRB5 allele, and 3) a haplotype carrying a DRB5^* 0101 allele without a DRB1 ^* 15 or ^* 16 allele. The evolutionary origins of these haplotypes were postulated based on their associations with the DRB6 pseudogene. Within HLA haplotypes which carry DRB3, a new DRB3 ^* 0205 allele and one unusual DRB3 association were identified. Finally, two new null DRB4 alleles are described: DRB4 ^* 0201N, which exhibits a deletion in the second exon, and a second allele, DRB4 ^* null, which lacks the second exon completely. Gene conversion-like events and variation in the number of functional genes through reciprocal recombination and inactivation contribute to the diversity observed in the second expressed HLA-DRB loci. Received: 2 November 1996 / Revised: 23 December 1996     

Identification of the nucleotide substitution that generates the fourth polymorphic site in human deoxyribonuclease I (DNase I)

In addition to the three polymorphic sites responsible for protein polymorphism, a new polymorphic site has been identified in intron 7 of the human deoxyribonuclease I (DNase I) gene. Three phenotypes were observed on single-strand conformational polymorphism analysis of a 266-bp polymerase chain reaction-amplified fragment containing exon 7 and part of intron 7 of the human DNase I gene. DNA sequencing analysis demonstrated that a C-G substitution occurred at position 1978 in intron 7. This substitution was confirmed by restriction fragment length polymorphism analysis, since a new Msp1 site is created by the substitution. Population and family studies showed that the inheritance of the genotypes for DNase I C1978G polymorphism is controlled by two codominant alleles, tentatively designated DNASE1*1978C and *1978G. The gene frequencies in a Japanese population were significantly different from those in a Caucasian (German) population. The C1978G polymorphism is in linkage disequilibrium with the common DNase I protein phenotypes 1, 1–2, and 2. Received: 20 March 1996 / Revised: 14 May 1996     

TNFB polymorphisms characterize three lineages of TNF region microsatellite haplotypes

 The human major histocompatibility complex (MHC) contains a variety of genes, many of which are highly polymorphic and of immunological importance. A database of MHC extended haplotypes was used to integrate experimental, cell line, and population data. Three alleles of the human TNF-beta (lymphotoxin-alpha) gene were identified, named TNFB ^*1SL, TNFB ^*2LL, and TNFB ^*1LS, each representing a different lineage in the evolution of TNF region haplotypes. Lower variability in the length of the associated microsatellite alleles indicates that ^*1SL characterizes the youngest of the three haplotype lineages. Microsatellite haplotypes in the two older lineages show evidence for a coevolution of alleles through concerted expansions. Genetic predispositions to high and low TNF-alpha (cachectin) responses seem to have evolved independently in more than one lineage. The literature data suggest different, or even opposite, associations concerning the regulation of TNF-alpha in macrophages and lymphoid cells. Microsatellite ud may be the most informative marker for studies of the associations of individual TNF region markers with secretion levels, immunity, and disease. Received: 10 December 1996 / Revised: 21 May 1997     

Absence of association between the Gly40→Ser mutation in the human glucagon receptor and Japanese patients with non-insulin-dependent diabetes mellitus or impaired glucose tolerance

We investigated whether a G¹²³→A mutation causing a Gly⁴⁰→Ser substitution in exon 2 of the human glucagon receptor gene, which has been reported to be associated with non-insulin-dependent diabetes mellitus (NIDDM) and impaired glucose tolerance (IGT) in France and Sardinia with a prevalences as high as 4.6% and 8.3%, respectively, is associated with Japanese patients with glucose intolerance. This mutation was not found in 242 unrelated Japanese patients with NIDDM or 23 with IGT by screening by the polymerase chain reaction-restriction fragment length polymorphism method. We also performed single-stranded conformational polymorphism analysis to search for new mutations in this gene associated with glucose intolerance. We found no mutations by examining all the 13 exons from 30 selected patients with NIDDM who had at least 2 diabetic first-degree relatives. These patients were also screened for the common polymorphism at codon 155 reported previously, but none were found to carry it. The absence of the mutation and polymorphism, which are common in French and Sardinian NIDDM or IGT patients, in Japanese indicates the existence of marked ethnic differences. Received: 19 February 1996     

Simultaneous identification of GSTP1 Ile105→Val105 and Ala114→Val114 substitutions using an amplification refractory mutation systempolymerase chain reactionassay: studies in patients with asthma

Background  

The glutathione S-transferase (GST) enzyme GSTP1 utilizes byproducts of oxidative stress. We previously showed that alleles of GSTP1 that encode the Ile105→Val105 substitution are associated with the asthma phenotypes of atopy and bronchial hyperresponsiveness (BHR). However, a further polymorphic site (Ala114→Val114) has been identified that results in the following alleles: GSTP1 ^* A (wild-type Ile105→Ala114), GSTP1 ^* B (Val105→Ala114), GSTP1 ^* C (Val105→Val114) and GSTP1 ^* D (Ile105→Val114).     

Polymorphisms in the TNF gene cluster and MHC serotypes in the West of Scotland

 We examined the distribution of polymorphic elements within the tumor necrosis factor (TNF) gene cluster in 105 unrelated individuals and determined their relationship to class I and class II major histocompatibility complex (MHC) antigens, and to the highly polymorphic microsatellites TNFa and TNFb. The data demonstrate the contribution of elements within the TNF cluster to two extended haplotypes which are recognized to straddle the MHC. The A1.B8.DR3 haplotype appears to contain the TNF alleles TNFa2, TNFb3, LT.Nco-1.B ^* 1, and TNF-308.2, while the A3.B7.DR2 haplotype is associated with the TNF alleles TNFa11, TNFb6, TNFc1, LT.Nco-1.B ^* 2, LT.AspH1.1, TNF-308.1, and TNFe1. The presence of other extended associations which covered smaller parts of the MHC was also suggested. In most cases, the associations described here were in keeping with previously described extended haplotypes which dominate the structure of the MHC, but these did not always match completely. Taken together, these data suggest that the structure of the TNF locus is well integrated into the rest of the MHC but that important ethnic differences may exist. Received: 12 June 1996 / Revised: 27 September 1996     

β-Thalassemia and βA globin gene haplotypes in Mexican mestizos

β-globin haplotypes of 20 β-thalassemia (β-thal) and 87 β^A Mexican mestizo chromosomes were analyzed to ascertain the origin of the β-thal alleles and the frequencies and distribution of the β^A haplotypes among northwestern Mexican mestizos. Sixteen β-thal chromosomes carried six Mediterranean alleles [five codon 39 C→T; two IVS1:1 G→A; two IVS1:5 G→A; three IVS1:110 G(A; one codon 11 (–T) and three (δβ)°-thal]; the remaining four were linked to three rare alleles (two –28 A→C and one each: –87 C→T and initiation codon ATG→GTG). Among the 87 β^A chromosomes, 17 different 5′ haplotypes with frequencies for 1, 3, 2 and 5 of 39.0%, 17. 2%, 9.2% and 6.9%, respectively, were observed. The β-haplotype analysis showed that 13 out of 16 Mediterranean chromosomes could easily be explained by gene migration; however, one codon 39 associated with haplotype 4 (– – – – + + –), one IVS1:1 with haplotype 1(+ – – – – + +) and one IVS1:5 G→A, may represent separate mutational events. Analysis of the rare alleles showed that the –28 A→C mutation was associated with the commonest β^A haplotype in Mexican mestizos, Mediterraneans and the total world population; therefore an independent origin cannot be ruled out. The –87 C→T and initiation codon ATG→GTG were found with β-haplotypes different from the reported ones, suggesting an indigenous origin. Received: 23 April 1996 / Revised: 10 September 1996     

Three new HLA-G alleles and their linkage disequilibria with HLA-A

Three new allelic forms of the HLA-G DNA sequence (HLA-G*II, HLA-G*III, and HLA-G*IV) have been identified. With the HLA-G*I sequence (previously designated HLA 6.0) as a reference, HLA-G*II shows a silent (G A) mutation at the third base of codon 57, HLA-G*III bears a non-synonymous (A T), but conservative, (Thr Ser) substitution at the first base of codon 31, and HLA-G*IV shows two silent substitutions: (A T) at the third base of codon 107 and (G A) at the third base of codon 57. A rapid method of singling out each allele on genomic DNA has been developed by using polymerase chain reaction amplification followed by restriction endonuclease treatment. Also, more or less strong linkage disequilibria has been found between most HLA-A alleles and either HLA-G*I or *II, both being the most prevalent alleles in the population, with a genotypic frequency of 0.55 and 0.38, respectively; HLA-G*III is very rare and HLA-G*IV has a genotypic frequency of 0.07. An evolutive classification of HLA-A alleles results according to their association with either HLA-G*I or HLA-G*II, which does not correlate with the classical serological cross-reacting groups classification. The finding of a strong and selective A/G linkage disequilibria with most HLA-A alleles, together with the existence of less frequent random A/G associations, may suggest that there exist in different haplotypes true and varied A/G genetic distances (and not a recombinational hotspot). It may be inferred from preliminary data that in primates HLA-A/G haplotypes bearing G*II may have appeared later than those bearing G*I.The nucleotide sequence data reported in this paper have been submitted to the GenBank and EMBL nucleotide sequence databases and have been assigned the following accession numbers: EMBL-X60983 (HLA-G*II), GenBank-M99048 (HLA-G*III), and GenBank-L07784 (HLA-G*IV).The contribution to this paper by P. Morales and A. Corell is equal, and the order of authorship is arbitrary. Correspondence to: A. Arnaiz-Villena.     

Molecular basis of the apolipoprotein H (β2-glycoprotein I) protein polymorphism

Apolipoprotein H (apoH, protein; APOH, gene) is considered to be an essential cofactor for the binding of certain antiphospholipid autoantibodies to anionic phospholipids. APOH exhibits a genetically determined structural polymorphism due to the presence of three common alleles (APOH*1, APOH*2 and APOH*3 ) detectable by isoelectric focusing (IEF) and immunoblotting. The APOH*3 allele can be further characterized into two subtypes, APOH*3^W and APOH*3^B, based upon its reactivity with monoclonal antibody 3D11. In this study we have determined the molecular basis of the APOH protein polymorphism and its distribution in three large U.S. population samples comprising 661 non-Hispanic whites, 444 Hispanics and 422 blacks. By direct DNA sequencing of PCR amplified fragments corresponding to the eight APOH exons, we identified two missense mutations that correspond to the APOH*1 and APOH*3^W alleles. A missense mutation (G→A) in exon 3, which alters amino acid Ser to Asn at codon 88 and creates a restriction site for TSP509 I, was present in all APOH*1 allele carriers. A second missense mutation (G→C) at codon 316 in exon 8, which replaces amino acid Trp with Ser and creates a restriction site for BSTBI, was present in all APOH*3^W carriers. The distribution of the Ser 88 Asn and Trp 316 Ser mutations was significantly different between the three racial groups. The frequency of the Asn-88 allele was 0.011, 0.043, and 0.056 in blacks, Hispanics and non-Hispanic whites, respectively. While the Ser-316 allele was observed sporadically in blacks (0.008), it was present at a polymorphic frequency in Hispanics (0.027) and non-Hispanic whites (0.059). The identification of the molecular basis of the APOH protein polymorphism will help to elucidate the structural – functional relationship of apoH in the production of antiphospholipid autoantibodies. Received: 20 November 1996 / Accepted: 13 February 1997     

Sequence-based association analysis of HLA class I and II alleles in Japanese supports conservation of common haplotypes

 Alleles of HLA-A, B, C, DRB1, DQB1, and DPB1 loci were fully determined in 117 healthy Japanese. A ^* 2402, A ^* 3303, A ^* 1101, A ^* 0201, B ^* 4403, B ^* 5201, Cw ^* 0102, Cw ^* 1403, Cw ^* 0304, Cw ^* 0702, Cw ^* 0801, and Cw ^* 1202 showed frequencies of over 10%. Multi-locus haplotype frequencies were estimated by the maximum likelihood method. Strength of association between C and B loci was comparable with that between DRB1 and DQB1 loci. Alleles unidentified by a serological method and having very similar nucleotide sequences (A2: A ^* 0201, A ^* 0206, A ^* 0207, B61: B ^* 4002, B ^* 4006) were carried by different haplotypes. Several frequent five-locus haplotypes were identified including A ^* 3303-Cw ^* 1403-B ^* 4403-DRB1 ^* 1302-DQB1 ^* 0604, and A ^* 2402-Cw ^* 1202-B ^* 5201-DRB1 ^* 1502-DQB1 ^* 0601. These sequence-based haplotypes corresponded to serology-based common haplotypes which have already been described in Japanese. These findings indicate that common HLA haplotypes consist of particular sets of HLA alleles and that these haplotypes have been conserved through recent human evolution. Received: 25 November 1996 / Revised: 20 January 1997     

Germline TCR-A restriction of immunoglobulin E responses to allergen

 Immunoglobulin E responses to known environmental antigens (allergens) may serve as a general model to investigate germline genetic restriction of the immune response. We have previously shown genetic linkage between IgE responses to major allergens and the T-cell receptor (TCR) A/D locus, but not to TCR-B, implying that elements in TCR A/D restrict the ability to react to specific antigens. We now show, in two sets of subjects from the same population, a strong allelic association between a VA8.1 polymorphism (VA8.1 ^* 2) and reactivity to Der p II, a major antigenic component of the house dust mite Dermatophagoides pteronyssinus. Association was also seen between Der p II IgE titres and HLA-DRB1 ^* 1501 alleles. Reactivity to Der p II was confined to subjects who were positive for VA8.1 ^* 2 and HLA-DRB1 ^* 1501, demonstrating germline HLA-DR and TCR-A interaction in restricting the response to exogenous antigen. Received: 28 January 1997 / Revised: 28 February 1997     

Two new mutations in the sterol 27-hydroxylase gene in two families lead to cerebrotendinous xanthomatosis

This report concerns two new mutations in the sterol 27-hydroxylase gene in two patients with cerebrotendinous xanthomatosis (CTX). In a Surinam-Creole patient (patient A), a G deletion on position cDNA 546/547 in exon 3 led to a frameshift and the introduction of a premature termination codon. In a Dutch patient (patient B), a C→T transition at position 496 in exon 3 also led to a premature termination codon. Patient A was homozygous for the mutation, whereas patient B was compound heterozygous, a C→T transition also being found in exon 6 at position 1204. The two new mutations were confirmed by restriction analysis with the restriction enzymes FokI and MaeI, respectively. Received: 24 July 1996 / Revised: 9 August 1996     

Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater

Denitrification of a high-strength synthetic wastewater (150 g NO^- ₃ l^-1) was carried out using a wine distillery effluent as an example of an industrial carbon source (22.7 g chemical oxygen demand l^-1). Two configurations were tested: one consisted of an acidogenesis reactor followed by a denitrifying reactor and the other was a single reactor directly fed with the raw effluents. In both cases, denitrification was achieved at a nitrate load of 9.54 g NO^- ₃ l^-1 day^-1 (2.19 g N as NO^- ₃ l^-1 day^-1) with good specific reduction rates: 32.6 mg and 35.2 mg N as NO _x  g volatile suspended solids h^-1, calculated on a single day, for the two-step and the one-step process respectively. Dissimilatory nitrate reduction to ammonium did not occur, even in the one-step process. Received: 26 October 1995/Received revision: 15 February 1996/Accepted: 20 February 1996     

Hb Costa Rica or · 2 ‚ 277(EF1)His→Arg: the first example of a somatic cell mutation in a globin gene

We have identified a minor hemoglobin component (∼5%) in the blood of a healthy Costa Rican female, but not in her mother and two brothers (father not studied), that has an His→Arg replacement at position β77 (Hb Costa Rica). No other amino acid replacements were observed and no β- or γ-chain-like peptides were present. Hb Costa Rica has a normal stability. Sequence analyses of numerous polymerase chain reaction (PCR)-amplified segments of DNA that contain exon 2 of the β gene failed to identify a CAC→CGC (His→Arg) mutation. The same was the case when cDNA was sequenced, indicating that a β-Costa Rica-mRNA could not be detected with this procedure. Gene mapping of genomic DNA with BglII, BamHI, and HindIII gave normal fragments only and with the same intensity as observed for the fragments of a normal control. The quantities of the β chain variants Hb J-Iran and Hb Fukuyama with related mutations at β77 vary between 30% and 45% in heterozygotes, whereas that of Hb F-Kennestone with the same His→Arg mutation but in the ^Gγ-globin gene, is a high 40%–45% (as percentage of total ^Gγ) in a heterozygous newborn. These different observations exclude a heterozygosity of the A→G mutation at codon β77, as well as a deletion comparable to that of Hbs Lepore or Kenya, or a β-globin gene duplication, and point to a nontraditional inheritance of Hb Costa Rica. Allele-specific amplification of cDNA with appropriate primers identified the presence of a low level of mutated mRNA in the reticulocytes of the patient, which was confirmed by dotblot analysis of the same material with ³²P-labeled probes. Comparable amplification products were not observed in genomic DNA. The A→G mutation apparently occurred in a somatic cell at a relatively early stage in the development of the hematopoietic cell system, and Hb Costa Rica accumulated through rapid cell divisions in patchy areas in the bone marrow (somatic mosaicism). An unequal distribution of Hb Costa Rica over the red cells supports this possibility. Received: 25 August 1995 / Revised: 13 December 1995