Genetic drift outweighs balancing selection in shaping post-bottleneck major histocompatibility complex variation in New Zealand robins (Petroicidae)

The Chatham Island black robin, Petroica traversi, is a highly inbred, endangered passerine with extremely low levels of variation at hypervariable neutral DNA markers. In this study we investigated variation in major histocompatibility complex (MHC) class II genes in both the black robin and its nonendangered relative, the South Island robin Petroica australis australis. Previous studies have shown that Petroica have at least four expressed class II B MHC genes. In this study, the sequences of introns flanking exon 2 of these loci were characterized to design primers for peptide-binding region (PBR) sequence analysis. Intron sequences were comprised of varying numbers of repeated units, with highly conserved regions immediately flanking exon 2. Polymerase chain reaction primers designed to this region amplified three or four sequences per black robin individual, and eight to 14 sequences per South Island robin individual. MHC genes are fitness-related genes thought to be under balancing selection, so they may be more likely to retain variation in bottlenecked populations. To test this, we compared MHC variation in the black robin with artificially bottlenecked populations of South Island robin, and with their respective source populations, using restriction fragment length polymorphism analyses and DNA sequencing of the PBR. Our results indicate that the black robin is monomorphic at class II B MHC loci, while both source and bottlenecked populations of South Island robin have retained moderate levels of variation. Comparison of MHC variation with minisatellite DNA variation indicates that genetic drift outweighs balancing selection in determining MHC diversity in the bottlenecked populations. However, balancing selection appears to influence MHC diversity over evolutionary timescales, and the effects of gene conversion are evident.     

Comparison of MHC genes among distantly related members of the genus Mus

The genomic content of class I and class II MHC DNA sequences in a variety of wild mice has been analyzed. The panel of mice includes members of three subgenera of the genus Mus. By genomic hybridization with the use of a variety of DNA probes, both class I and class II DNA sequences appear to be conserved in all of the species examined. However, the number of class I DNA sequences differs among the species. Furthermore, this variation appears to result from differential increases within subsets of class I genes. These data suggest that the class I multigene family is dynamic and changing over short periods of evolutionary time. In contrast, none of the class II genes appears to vary in copy number. More extensive polymorphism was noted amongst the class II beta genes than the alpha genes. Interestingly, the genomic sequence corresponding to E beta 2 is highly conserved, leading to the prediction that it is a genetically functional sequence.     

Diversity and rearrangement of the human T cell rearranging gamma genes: nine germ-line variable genes belonging to two subgroups

We describe nine T cell gamma variable (V) gene segments isolated from human DNA. These genes, which fall into two subgroups, are mapped in two DNA regions covering 54 kb and probably represent the majority of human V gamma genes. One subgroup (V gamma I) contains eight genes, consisting of four active genes and four pseudogenes. The single V gamma II gene is potentially active. Sequence analysis of the V gamma I genes shows variation clustered in hypervariable regions, but somatic variability is restricted to N-region diversity. Studies on rearrangement in T cell lines and in thymic DNA show that major rearrangements can be observed that are attributable to the five active V gamma genes. In addition, human cells with the phenotype of helper T cells can undergo productive V gamma-J gamma joining.     

High levels of genetic variation at MHC class II DBB loci in the tammar wallaby (<Emphasis Type="Italic">Macropus eugenii</Emphasis>)

High levels of MHC diversity are crucial for immunological fitness of populations, with island populations particularly susceptible to loss of genetic diversity. In this study, the level of MHC class II DBB diversity was examined in tammar wallabies (Macropus eugenii) from Kangaroo Island by genotyping class II-linked microsatellite loci and sequencing of DBB genes. Here we show that the tammar wallaby has at least four expressed MHC class II DBB loci and extensive genetic variation in the peptide-binding region of the DBB genes. These results contradict early studies which suggested that wallabies lacked MHC class II diversity and demonstrate that, in spite of the long-term isolation on an offshore island, this population of wallabies has a high level of DBB diversity.     

Fugu orthologues of human major histocompatibility complex genes: a genome survey

The major histocompatibility complex (MHC) region in fish has been subjected to piecemeal analysis centering on the in-depth characterization of single genes. The emphasis has been on those genes proven to be involved in the immune response such as the class I and class II antigen presenting genes and the complement genes. The Fugu genome data presents the opportunity to examine the short-range linkage of potentially all the human MHC orthologues and examine conserved synteny with the human and, to a more limited extent, zebrafish genomes. Analysis confirms the existence of a limited MHC locus in Fugu comprising the MHC class Ia genes and associated class II region genes involved in class I antigen presentation. Identification of additional human MHC orthologues indicates the completely dispersed nature of this region in fish, with a maximum of six MHC genes maintained within close proximity in any one contig. The majority of the other genes are present in the genome data as either singletons or pairs. Comparison with zebrafish substantiates previously observed linkages between class III region orthologues and hints at an ancient conserved class III region.     

Analysis of a horse family with a crossing-over between the ELA complex and the A blood group system

A horse family in which a recombination occurred in the chromosome region coding for the serological specificities of the ELA complex and those of the A blood group system of a mare was further analysed by mixed lymphocyte reaction (MLR) and Southern blot hybridization. This family consisted of a stallion, a mare and five full sibs. The stallion and the mare were heterozygous for internationally recognized ELA specificities while only the mare was heterozygous for the A blood group system. MLR between all members of the family confirmed that the stallion possessed two different ELA haplotypes and suggested that recombination in the mare occurred outside the segment delimited by the ELA-A locus and the MLR region. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI), three human HLA probes (one of class I cDNA and two of class II probes), one cDNA (DR beta) and one genomic (DQ alpha). Class I and class II restriction fragments of the mare segregated in accordance to the ELA specificities and thus clearly confirming that the crossing-over did not occur between the ELA-A gene and the class I, class II region nor between DR beta and DQ alpha subsets. The A blood group genetic determinants would thus be situated outside the ELA region defined by class I and class II genes.     

MHC class I loci of the Bar-Headed goose (Anser indicus)

MHC class I proteins mediate functions in anti-pathogen defense. MHC diversity has already been investigated by many studies in model avian species, but here we chose the bar-headed goose, a worldwide migrant bird, as a non-model avian species. Sequences from exons encoding the peptide-binding region (PBR) of MHC class I molecules were isolated from liver genomic DNA, to investigate variation in these genes. These are the first MHC class I partial sequences of the bar-headed goose to be reported. A preliminary analysis suggests the presence of at least four MHC class I genes, which share great similarity with those of the goose and duck. A phylogenetic analysis of bar-headed goose, goose and duck MHC class I sequences using the NJ method supports the idea that they all cluster within the anseriforms clade.     

Locus specificity of polymorphic alleles and evolution by a birth-and-death process in mammalian MHC genes

We have conducted an extensive phylogenetic analysis of polymorphic alleles from human and mouse major histocompatibility complex (MHC) class I and class II genes. The phylogenetic tree obtained for 212 complete human class I allele sequences (HLA-A, -B, and -C) has shown that all alleles from the same locus form a single cluster, which is highly supported by bootstrap values, except for one HLA-B allele (HLA-B*7301). Mouse MHC class I loci did not show locus-specific clusters of polymorphic alleles. This was considered to be because of either interlocus genetic exchange or the confusing designation of loci in different haplotypes at the present time. The locus specificity of polymorphic alleles was also observed in human and mouse MHC class II loci. It was therefore concluded that interlocus recombination or gene conversion is not very important for generating MHC diversity, with a possible exception of mouse class I loci. According to the phylogenetic trees of complete coding sequences, we classified human MHC class I (HLA-A, -B, and -C) and class II (DRB1) alleles into three to five major allelic lineages (groups), which were monophyletic with high bootstrap values. Most of these allelic groups remained unchanged even in phylogenetic trees based on individual exons, though this does not exclude the possibility of intralocus recombination involving short DNA segments. These results, together with the previous observation that MHC loci are subject to frequent duplication and deletion, as well as to balancing selection, indicate that MHC evolution in mammals is in agreement with the birth-and-death model of evolution, rather than with the model of concerted evolution.     

Gene organisation, sequence variation and isochore structure at the centromeric boundary of the human MHC.

We have mapped and sequenced the region immediately centromeric of the human major histocompatibility complex (MHC). A cluster of 13 genes/pseudogenes was identified in a 175 kb PAC linking the TAPASIN locus with the class II region. It includes two novel human genes (BING4 and SACM2L) and a thus far unnoticed human leucocyte antigen (HLA) class II pseudogene, termed HLA-DPA3. Analysis of the G+C content revealed an isochore boundary which, together with the previously reported telomeric boundary, defines the MHC class II region as one of the first completely sequenced isochores in the human genome. Comparison of the sequence with limited sequence from other cell lines shows that the high sequence variation found within the classical class II region extends beyond the identified isochore boundary leading us to propose the concept of an "extended MHC". By comparative analysis, we have precisely identified the mouse/human synteny breakpoint at the centromeric end of the extended MHC class II region between the genes HSET and PHF1.     

Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.     

Development of a high-resolution NGS-based HLA-typing and analysis pipeline

The human leukocyte antigen (HLA) complex contains the most polymorphic genes in the human genome. The classical HLA class I and II genes define the specificity of adaptive immune responses. Genetic variation at the HLA genes is associated with susceptibility to autoimmune and infectious diseases and plays a major role in transplantation medicine and immunology. Currently, the HLA genes are characterized using Sanger- or next-generation sequencing (NGS) of a limited amplicon repertoire or labeled oligonucleotides for allele-specific sequences. High-quality NGS-based methods are in proprietary use and not publicly available. Here, we introduce the first highly automated open-kit/open-source HLA-typing method for NGS. The method employs in-solution targeted capturing of the classical class I (HLA-A, HLA-B, HLA-C) and class II HLA genes (HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1). The calling algorithm allows for highly confident allele-calling to three-field resolution (cDNA nucleotide variants). The method was validated on 357 commercially available DNA samples with known HLA alleles obtained by classical typing. Our results showed on average an accurate allele call rate of 0.99 in a fully automated manner, identifying also errors in the reference data. Finally, our method provides the flexibility to add further enrichment target regions.     

Haploinsufficiency of DNA Damage Response Genes and their Potential Influence in Human Genomic Disorders

Genomic disorders are a clinically diverse group of conditions caused by gain, loss or re-orientation of a genomic region containing dosage-sensitive genes. One class of genomic disorder is caused by hemizygous deletions resulting in haploinsufficiency of a single or, more usually, several genes. For example, the heterozygous contiguous gene deletion on chromosome 22q11.2 causing DiGeorge syndrome involves at least 20-30 genes. Determining how the copy number variation (CNV) affects human variation and contributes to the aetiology and progression of various genomic disorders represents important questions for the future. Here, I will discuss the functional significance of one form of CNV, haploinsufficiency (i.e. loss of a gene copy), of DNA damage response components and its association with certain genomic disorders. There is increasing evidence that haploinsufficiency for certain genes encoding key players in the cells response to DNA damage, particularly those of the Ataxia Telangiectasia and Rad3-related (ATR)-pathway, has a functional impact. I will review this evidence and present examples of some well known clinically similar genomic disorders that have recently been shown to be defective in the ATR-dependent DNA damage response. Finally, I will discuss the potential implications of a haploinsufficiency-induced defective DNA damage response for the clinical management of certain human genomic disorders.Key Words: DNA damage response, ATR, haploinsufficiency, genomic disorders.     

Evolution of the Mhc class I region: the framework hypothesis

 A comparison of the major histocompatibility complex (Mhc) region between human and mouse highlights both stability and differences. The class II and class III regions are orthologous; they probably existed in the ancestor in a similar organization and were not subjected to major rearrangement. The class I genes, by contrast, are definitely paralogous, having been reorganized several times. As long as only class I genes were identified, the class I regions of human and mouse were difficult to compare directly. The identification of non-class I genes has allowed a comparative map to be drawn, which shows that the class I region is orthologous between human and mouse as well. The lack of orthology specifically applies to the class I sequences. However, the comparative map shows that the non-orthologous class I sequences occupy homologous locations with regard to the conserved genes. I propose a model to explain this paradox. The conserved genes may represent samples of a dense "framework" of genes whose alterations are deleterious. The homologous positions occupied by class I genes would thus represent the few permissive places allowing major perturbations. The evolution of the class I sequences, by duplication and deletion, independently in the two species, has taken place within the scope defined by the framework: insertion at the permissive places, and expansion by creation of class I-related DNA by duplication, thus pushing back the boundaries of the framework. Received: 23 March 1998 / Revised: August 14 1998     

Sequence of the human CALM II calmodulin gene promoter region

A clone containing a portion of the promoter region for the human bona fide CALM II gene was isolated using a human Promoter Finder DNA Walking Kit. This promoter region contains, putatively, a GC box (common in housekeeping genes), a CRE-binding site, a TATA like box and AGGGA sequences. The latter are reported to be present in genes for Ca2+ binding genes. This human promoter region exhibits overall 85% sequence identity to the corresponding region of the rat CALM II promoter but shows no identity to the corresponding region of the human CALM I or CALM III promoters.     

First domain encoding sequence mediates human class II beta-chain gene cross-hybridization

HLA class II molecules are surface heterodimers which are essential in the initiation of immune responses. The amount of polymorphism expressed by the different class II molecules is largely dependent on the polymorphic structure of their beta chains. Cross-hybridization between, class II beta genes frequently hampered restriction fragment length polymorphism analysis of donor genomic DNA. In this report we show that the cross-hybridization between human class II beta genes is mediated by a region of high homology, rich in C and G residues, between the first domain encoding sequences of DP, DQ, and DR genes. The removal of the DNA segment containing this region from the fragments used as labeled probes against the corresponding fragments of the genes at other loci or against endonuclease digested genomic DNA completely eliminated or drastically reduced the cross-hybridization. Also, the RFLP patterns generated with the shortened probes were more informative and much simpler to interpret than were these generated with probes made from the original genes.     

Molecular characterization and genetic mapping of class I and class II MHC genes of the domestic cat

The major histocompatibility complex (MHC) of the domestic cat has been poorly characterized to date, primarily because of numerous difficulties in the preparation of allotypic sera. We present here a comparative analysis of class I and class II genes in domestic cat populations using molecular probes of the MHC from man and mouse. The cat possesses a minimum of 20 class I loci and 5 class II genes per haploid genome. Class I genes of the domestic cat expressed limited restriction fragment length polymorphism. The average percent difference of the size of DNA fragments between individual cats was 9.0 %, a value five times lower than the value for mice, but comparable to the human DNA polymorphism level. Class I and class II genes were both genetically mapped to feline chromosome B2 using a panel of rodent x cat somatic cell hybrids. Since feline chromosome B2 is syntenically homologous to human chromosome 6 and mouse chromosome 17, these results affirm the linkage conservation of the MHC-containing linkage group in the three mammalian orders.     

Genetic variability between two breeds based on restriction fragment length polymorphisms (RFLPs) of major histocompatibility complex class I genes in the pig

Summary Restriction fragment length polymorphism analyses of SLA class I genes were performed on 55 Duroc and 24 Hampshire boars from the 1986–87 national performance tests of each breed. Few boars were inbred. Southern blotting and hybridization procedures were performed on genomic DNA isolated from white blood cells by using Pvu II, Bam HI, and Eco RI endonucleases and a swine MHC class I probe. Genetic variability within and between the two breeds was estimated in terms of nucleotide diversity, by using a mathematical analysis based on the different RFLP patterns. The nucleotide diversity calculated within each breed was less than that between the two breeds. The results from the nucleotide diversity analysis suggested that genetic variability was greater in the Duroc breed than in the Hampshire breed. A relatively high level of genetic variability was shown in the class I major histocompatibility complex genes in the pig.     

Comparative and evolutionary analysis of the rhesus macaque extended MHC class II region

The sequence-based map of a part of the rhesus macaque major histocompatibility complex (MHC) extended class II region is presented. The sequenced region encompasses 67,401 bp and contains the SACM2L, RING1, FABGL and KE4 genes, as well as the HTATSF1-like and ZNF-like pseudogenes. Similar to human, but different from rat and mouse, no class I genes are found in the SACM2L- RING1 interval. The rhesus macaque extended MHC class II region shows a high degree of conservation of exonic as well as intronic and intergenic sequences compared with the respective human region. It is concluded that this particular genomic organization of the extended class II region-i.e., the absence of class I genes and the presence of the HTATSF1-like and ZNF-like pseudogenes-can be traced back to a common ancestor of humans and rhesus macaques about 23 million years ago.