<Emphasis Type="Italic">IGHV1</Emphasis>,<Emphasis Type="Italic"> IGHV5</Emphasis> and<Emphasis Type="Italic"> IGHV7</Emphasis> subgroup genes in the Rhesus macaque

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.     

Genomic screening by 454 pyrosequencing identifies a new human IGHV gene and sixteen other new IGHV allelic variants

Complete and accurate knowledge of the genes and allelic variants of the human immunoglobulin gene loci is critical for studies of B cell repertoire development and somatic point mutation, but evidence from studies of VDJ rearrangements suggests that our knowledge of the available immunoglobulin gene repertoire is far from complete. The reported repertoire has changed little over the last 15 years. This is, in part, a consequence of the inefficiencies involved in searching for new members of large, multigenic gene families by cloning and sequencing. The advent of high-throughput sequencing provides a new avenue by which the germline repertoire can be explored. In this report, we describe pyrosequencing studies of the heavy chain IGHV1, IGHV3 and IGHV4 gene subgroups in ten Papua New Guineans. Thousands of 454 reads aligned with complete identity to 51 previously reported functional IGHV genes and allelic variants. A new gene, IGHV3-NL1*01, was identified, which differs from the nearest previously reported gene by 15 nucleotides. Sixteen new IGHV alleles were also identified, 15 of which varied from previously reported functional IGHV genes by between one and four nucleotides, while one sequence appears to be a functional variant of the pseudogene IGHV3-25. BLAST searches suggest that at least six of these new genes are carried within the relatively well-studied populations of North America, Europe or Asia. This study substantially expands the known immunoglobulin gene repertoire and demonstrates that genetic variation of immunoglobulin genes can now be efficiently explored in different human populations using high-throughput pyrosequencing.     

Many human immunoglobulin heavy-chain IGHV gene polymorphisms have been reported in error

The identification of the genes that make up rearranged immunoglobulin genes is critical to many studies. For example, the enumeration of mutations in immunoglobulin genes is important for the prognosis of chronic lymphocytic leukemia, and this requires the accurate identification of the germline genes from which a particular sequence is derived. The immunoglobulin heavy-chain variable (IGHV) gene repertoire is generally considered to be highly polymorphic. In this report, we describe a bioinformatic analysis of germline and rearranged immunoglobulin gene sequences which casts doubt on the existence of a substantial proportion of reported germline polymorphisms. We report a five-level classification system for IGHV genes, which indicates the likelihood that the genes have been reported accurately. The classification scheme also reflects the likelihood that germline genes could be incorrectly identified in mutated VDJ rearrangements, because of similarities to other alleles. Of the 226 IGHV alleles that have previously been reported, our analysis suggests that 104 of these alleles almost certainly include sequence errors, and should be removed from the available repertoire. The analysis also highlights the presence of common mismatches, with respect to the germline, in many rearranged heavy-chain sequences, suggesting the existence of twelve previously unreported alleles. Sequencing of IGHV genes from six individuals in this study confirmed the existence of three of these alleles, which we designate IGHV3-49*04, IGHV3-49*05 and IGHV4-39*07. We therefore present a revised repertoire of expressed IGHV genes, which should substantially improve the accuracy of immunoglobulin gene analysis.     

On the genomics of immunoglobulins in the gray,short-tailed opossum <Emphasis Type="Italic">Monodelphis domestica</Emphasis>

Annotated maps of the IGH, IGK, and IGL loci in the gray, short-tailed opossum Monodelphis domestica were generated from analyses of the available whole genome sequence for this species. Analyses of their content and organization confirmed a number of previous conclusions based on characterization of complementary DNAs encoding opossum immunoglobulin heavy and light chains and limited genomic analysis, including (a) the predominance of a single immunoglobulin heavy chain variable region (IGHV) subgroup and clan, (b) the presence of a single immunoglobulin (Ig)G subclass, (c) the apparent absence of an IgD, and (d) the general organization and V gene complexity of the IGK and IGL light chain loci. In addition, several unexpected discoveries were made including the presence of a partial V to D, germline-joined IGHV segment, the first germline-joined Ig V gene to be found in a mammal. In addition was the presence of a larger number of IGKV subgroups than had been previously identified. With this report, annotated maps of the major histocompatibility complex, T-cell receptor, and immunoglobulin loci have been completed for M. domestica, the only non-eutherian mammalian species for which this has been accomplished, strengthening the utility of this species as a model organism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat

We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species.     

IGH V3-23*01 and its allele V3-23*03 differ in their capacity to form the canonical human antibody combining site specific for the capsular polysaccharide of Haemophilus influenzae type b

The IGH V3-23*01 gene is used in the formation of the canonical combining site which dominates the human antibody repertoire to the Haemophilus influenzae type b (Hib) polysaccharide (PS). An allele of the human IGH V3-23*01 gene, known as V3-23*03, differs from V3-23*01 in nine bases, eight of which are located in the second complementarity determining region. These eight differences encode five amino acid substitutions. In this study we investigated whether the V3-23*03 sequence polymorphism affected Hib PS binding. We constructed two Fab fragments that had the canonical Hib PS combining site VH-VL configuration but that had either V3-23*01 or V3-23*03. Radioantigen binding assay showed that on a concentration basis the V3-23*03 Fab was 20-fold more effective in binding Hib PS than the V3-23*01 Fab. The V3-23*03 Fab was 4-fold more effective than the V3-23*01 Fab in mediating facilitated bactericidal activity against Hib organisms. These findings identify a functional consequence of V3-23 allelism, and suggest that utilization of the V3-23*03 gene in the human Hib PS repertoire would generate canonical antibodies with higher affinity and protective efficacy than canonical antibodies utilizing V3-23*01. Thus, IGH V gene allelic variation has the potential to impact the generation of protective immunity to Hib.     

The investigation of allele and genotype frequencies of <Emphasis Type="Italic">CYP3A5 (1*/3*)</Emphasis> and <Emphasis Type="Italic">P2Y12</Emphasis> (T744C) in Iran

Research on the frequency of the highly functional mutations of genes coding required for metabolizing enzymes has shown significant ethnic variations. However, few studies, if any, have examined the frequency distribution of major allelic variations in the context of Iran. In this regard, the present study focused on the genotype profile of Southern Iranians in order to compare allele frequencies of their CYP3A5 and P2Y12 (T744C) which have been shown to have roles in metabolizing clopidogrel, with those of other populations. Therefore, genotyping was carried out on 112 unrelated individuals by PCR–RFLP. The CYP3A5*3 allele was found in 185 persons with allelic frequency 0.82, which is the most common allele among Caucasians (90–95%). The frequency of 82% is different from other Caucasians (90–94%), Indians (67%), Vietnam (67%) and Africans (15%). but lower than frequency in Chinese populations (74%) and Korean (76%). The allele frequency of the −744T (4%) is different from frequencies of Caucasian, American, Chinese, Korean, and Subsahara population. This study confirmed significant inter-ethnic differences in CYP3A5 and P2Y12 frequencies between Iranians and other ethnic groups. The results of this study will be useful for clinical pharmacokinetic investigations and drug dosage recommendations especially antiplatelet drugs such as Clopidogrel, for Iranians.     

Genetic Heterogeneity in the Indian Mus musculus

This study deals with the characterization of 10populations of M. musculus from differentgeographical locations in India. The genetics of Indianwild mice has been completely obscure and this is thefirst report on allozyme variations in the naturalpopulation. We have used a set of 24 biochemical geneticmarkers to measure levels of diversity within and amongpopulations. The allelic frequency data indicate extreme genetic variability, which is furtherenhanced by the presence of novel alleles. Overall thespecies shows a high level of heterogenity. The highlypolymorphic central populations of M. musculus cannot be assigned to any one particularsubspecies. The allelic profiles, however, indicate agradual differentiation toward the castaneus andbatcrianus subspecies lineages.     

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human V_H repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the V_H3 (41%), V_H4 (39%), and V_H1 (14%) subgroups used more frequently than the V_H5 (3.9%) or V_H7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human V_H genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.     

Evolutionary relationship ofHLA-DRB genes inferred from intron sequences

The major histocompatibility complex (Mhc) consists of class I and class II genes. In the humanMhc (HLA) class II genes, nineDRB loci have been identified. To elucidate the origin of these duplicated loci and allelic divergences at the most polymorphicDRBI locus, introns 4 and 5 as well as the 3′ untranslated region (altogether approximately 1,000 base pairs) of sevenHLA-DRB loci, threeHLA-DRBI alleles, and nine nonhuman primateDRB genes were examined. It is shown that there were two major diversification events inHLA-DRB genes, each involving gene duplications and allelic divergences. Approximately 50 million years (my) ago,DRBI ^*04 and an ancestor of theDRB1 ^*03 cluster (DRBI ^*03, DRBI^*15, andDRB3) diverged from each other andDRB5, DRB7, DRB8, and an ancestor of theDRB2 cluster (DRB2, DRB4, andDRB6) arose by gene duplication. Later, about 25 my ago,DRBI ^*15 diverged fromDRBI*03, andDRB3 was duplicated fromDRBI ^*03. Then, some 20 my ago, the lineage leading to theDRB2 cluster produced two new loci,DRB4 andDRB6. TheDRBI ^*03 andDRBI ^*04 allelic lineages are extraordinarily old and have persisted longer than some duplicated genes. The orthologous relationships ofDRB genes between human and Old World monkeys are apparent, but those between Catarrhini and New World monkeys are equivocal because of a rather rapid expansion and contraction of primateDRB genes by duplication and deletion. Correspondence to: Y. Satta     

An in vivo pilot study characterizing the new CYP2A6*7, *8, and *10 alleles.

We developed genotyping assays for CYP2A6*7 (Ile471Thr) and CYP2A6*8 (Arg485Leu). We found higher allelic frequencies in Japanese and Chinese versus Caucasians and identified an allele in which both substitutions occur together (CYP2A6*10). We created a homology model for predicting the impact of allelic variants on enzymatic activity and subsequently tested this in vivo in a pilot kinetic study. Consistent with our homology model predictions, we found (i) that CYP2A6*7 produces an enzyme that has decreased (not inactive) activity for metabolizing nicotine and coumarin; (ii) that CYP2A6*8 is unlikely to affect catalytic activity in vivo; and (iii) that having both substitutions together on an allele (CYP2A6*10) dramatically reduces function and may be fully inactive for some substrates. In conclusion, this study identifies, at relatively high frequency in Asians, an allele with decreased activity (may be substrate selective), a fully functional allele, and an allele containing both substitutions in which function is dramatically reduced.     

Genetics of esterase isoenzymes in Malus

Summary Three main zones of esterase activity (EST-I, EST-III, EST-IV) identified in leaf extracts of cultivated apple and Malus species were determined by the genes EST-1, EST-3 and EST-4, respectively. In addition to earlier reported alleles of EST-1 (a, b) three further bands c, d and f were identified in the EST-I zone of which c was found to be determined by an allele, c. Two alleles, a, b, and a null allele were found for both the genes EST-3 and EST-4. Differences in allelic frequency were observed between cultivars, rootstocks and Malus species. Allele EST-1a was rare amongst the rootstocks. The examination of Malus species and derivatives showed a geographical relationship. Allele EST-1c was confined to species of Asian origin, and EST-1d was confined to American species.     

A new<Emphasis Type="Italic"> DR7-DQ8</Emphasis> haplotype resulting from a recombination between the<Emphasis Type="Italic"> DQA1</Emphasis> and<Emphasis Type="Italic"> DQB1</Emphasis> loci in a leukemic patient of Caucasoid origin

Meiotic recombinations within the HLA-DR/DQ subregion are seldomly observed. However the high number of unusual DRB1-DQB1 allelic combinations underline the importance of crossover in shaping the class II haplotypic diversity. We present here the first report of a DQA1-DQB1 recombination event in a leukemic patient as detected by complete class II molecular typing of the family, including analysis of the DQCAR microsatellite. The recombination that occurred on the maternal chromosomes led to the unusual DR7-DQ8 haplotype characterized by the DRB1*0701-DRB4*01030102N-DQA1*0201-DQB1*0302 alleles. Because the patient had no HLA-identical sibling donor, a search for an unrelated hematopoietic stem cell donor was initiated. Out of three potential donors, only one HLA-A/-B/-C/DRB1-compatible but DQB1-mismatched donor could be identified.     

Allele frequency distribution of CYP2C9*2 and CYP2C9*3 polymorphisms in six Mexican populations

Allele frequency differences of functional CYP2C9 polymorphisms are responsible for some of the variation in drug response observed in human populations. The most relevant CYP2C9 functional variants are CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910). These polymorphisms show variation in allele frequencies among different population groups. The present study aimed to analyze these polymorphisms in 947 Mexican-Mestizo from Mexico City and 483 individuals from five indigenous Mexican populations: Nahua, Teenek, Tarahumara, Purepecha and Huichol. The CYP2C9*2 allele frequencies in the Mestizo, Nahua and Teenek populations were 0.051, 0.007 and 0.005, respectively. As for CYP2C9*3, the allelic frequencies in the Mestizo, Nahua and Teenek populations were 0.04, 0.005 and 0.005, respectively. The CYP2C9*2 and CYP2C9*3 alleles were not observed in the Tarahumara, Purepecha and Huichol populations. These findings are in agreement with previous studies reporting very low allele frequencies for these polymorphisms in American Indigenous populations.     

A triose-phosphate isomerase polymorphism in the Atlantic salmonSalmo salar L.

Atlantic salmon,Salmo salar L., from four European locations show allelic variation at one of three triose-phosphate isomerase (TPI) loci (TPI-3*) when separated on horizontal starch gel electrophoresis, using either eye or liver extracts. Two common alleles (*100 and*103) and one rare allele (*97) segregate atTPI-3* with unambiguous typing being possible by observing the interlocus heterodimers. Family studies demonstrate thatTPI-3* 100 and*103 are of autosomal location and are inherited in a Mendelian fashion.TPI-3* variation can also be typed in adipose fin tissue, allowing nondestructive tissue sampling. Three loci are also active in brown trout,Salmo trutta, with two individuals being homozygous forTPI-3*, as are a small number ofS. salar from eastern Canada. The presence of this additional variable allozyme locus inS. salar is important, since genetic studies in that species have been limited by the low level of allozyme variability detectable.     

Polymorphic SVA retrotransposons at four loci and their association with classical HLA class I alleles in Japanese, Caucasians and African Americans

Polymorphic insertion frequencies of the retrotransposons known as the “SVA” elements were investigated at four loci in the MHC class I genomic region to determine their allele and haplotype frequencies and associations with the HLA-A, -B or -C genes for 100 Japanese, 100 African Americans, 174 Australian Caucasians and 66 reference cell lines obtained from different ethnic groups. The SVA insertions representing different subfamily members varied in frequency between none for SVA-HF in Japanese and 65% for SVA-HB in Caucasians or African Americans with significant differences in frequencies between the three populations at least at three loci. The SVA loci were in Hardy–Weinberg equilibrium except for the SVA-HA locus which deviated significantly in African Americans and Caucasians possibly because of a genomic deletion of this locus in individuals with the HLA-A*24 allele. Strong linkage disequilibria and high percentage associations between the human leucocyte antigen (HLA) class I gene alleles and some of the SVA insertions were detected in all three populations in spite of significant frequency differences for the SVA and HLA class I alleles between the three populations. The highest percentage associations (>86%) were between SVA-HB and HLA-B*08, -B*27, -B*37 to -B*41, -B*52 and -B*53; SVA-HC and HLA-B*07; SVA-HA and HLA-A*03, -A*11 and -A*30; and SVA-HF and HLA-A*03 and HLA-B*47. From pairwise associations in the three populations and the homozygous cell line results, it was possible to deduce the SVA and HLA class I allelic combinations (haplotypes), population differences and the identity by descent of several common HLA-A allelic lineages.     

Allelism within the DEX and STA gene families in Saccharomyces diastaticus

Summary Saccharomyces diastaticus produces an extracellular glucoamylase and is therefore capable of hydrolyzing and fermenting starch. Tamaki (1978) studied starch utilization in S. diastaticus and found three polymeric genes controlling this function: STA1, STA2 and STA3. Independently, Erratt and Stewart (1978) studied dextrin utilization by the yeast S. diastaticus and designated the gene, which they identified, DEX1. Erratt and Stewart (1981a, b) later described two other genes which controlled glucoamylase production in S. diastaticus: DEX2 and a third which was allelic to STA3. At that time STA1 and STA2 were not available to test for allelism in the DEX gene family. In this study strains containing the remaining 4 genes have been examined to determine if further allelism exists between the two gene families. It was ascertained that DEX1 is allelic to STA2 and DEX2 is allelic to STA1. Therefore, no new gene controlling starch utilization has been identified and these two nomenclatures can now be consolidated into one. Based on the fact that the glucoamylase from S. diastaticus can hydrolyze both dextrin and starch, dextrin being the term used to described partially hydrolyzed starch, and the more wide use of the nomenclature STA, we propose to retain STA as the designation for genes coding for glucoamylase production in S. diastaticus.     

Targeted Ds-tagging strategy generates high allelic diversity at the Arabidopsis HY2 locus

The targeted (or directed) tagging is a strategy aimed to mobilize a tranposon into a specific gene (target). Only a very few Arabidopsis genes have been tagged by this way, thus the efficiency of the strategy, as well as the diversity of the alleles obtained are not well documented. We have used a maize Ds element in a directed tagging of HY2. The starting Ds element, located 22kb proximal to HY2, has been remobilized in a cross with an Ac transposase source line. From the F2 progeny of 4800 F1 we phenotypically isolated seven hy2 mutants. Molecular analysis of these alleles revealed that two contained a Ds element in HY2 and were instable, three have a large deletion that partially or completely removed HY2, one has a footprint in a HY2 exon and one leaky allele consisted of a 22 kb inversion upstream the HY2 coding sequence. Thus, the transposon-based directed tagging strategy generates a wide diversity of tagged and non-tagged alleles that can be used to generate allelic series or deletion of clustered genes.