# Patent

## Title
Means and methods for haplotyping MHC-DRB loci in mammals and uses thereof

## Abstract

The invention relates to the typing of MHC-DRB loci in mammals. In particular, the invention provides a typing procedure for the mammalian DRB region that allows an easy, economical, high resolution, fast and accurate haplotyping protocol. The invention further provides the use of said typing procedure in genetic applications, and provides a kit for typing of MHC-DRB loci.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. patent application Ser. No. 12/595,292, filed Jan. 7, 2010, which is the U.S. National Phase of, and Applicants claim priority from, International Application Number PCT/NL2008/050199, filed 10 Apr. 2008, European Patent Application No. 07105879.6, filed 10 Apr. 2007, and European Patent Application No. 07107288.8, filed 1 May 2007, each of which is incorporated herein by reference in its entirety.
The invention relates to the field of biology and medicine. In particular, the invention relates to the typing of MHC-DRB loci in mammals.
BACKGROUND OF THE INVENTION
The Major Histocompatibility Complex (MHC) is a large genomic region that is present in most vertebrates, although individual MHC region-linked genes have been identified in invertebrate genomes such as Drosophila melanogaster and Caenorhabditis elegans. The MHC gene products play a key role in adaptive immunology. Genes of the MHC are among the most polymorphic loci known in vertebrates, due to a high rate of mutation and recombination. Major differences between species reside not only in the number and identity of MHC genes, but also in the degree of allelic polymorphism for individual genes.
In general, three subgroups of MHC molecules can be defined, termed class I, II and III gene products. Molecules encoded by the so-called classical MHC class I and class II genes, present short peptides derived from proteins of pathogens and self antigens to the immune system. MHC class I genes encode molecules that are present on the surfaces of nearly all nucleated cells. These gene products are mainly responsible for facilitating immune responses to intracellularly processed pathogens such as viruses. MHC class II gene products are mainly present in a subset of antigen-presenting cells such as B-lymphocytes and activated T-lymphocytes. Class II gene products are primarily involved in the immune response against extracellular pathogens such as bacteria. Furthermore, they play an important role in recognition of foreign and self-antigens. MHC class III gene products are not involved in antigen presentation but rather represent other immune-related components such as components of the complement system.
Of particular importance are the MHC genes that encode the cell surface, antigen-presenting molecules. In humans, these genes are referred to as Human Leukocyte Antigen (HLA) genes. The classical MHC genes HLA-A, HLA-B, and HLA-C belong to MHC class I, encoding the alpha chain of the respective MHC molecule. The HLA class II region is divided into -DP, -DQ, and -DR. The classical HLA-DR, -DQ, and -DP molecules are transmembrane heterodimers, composed of an alpha- and beta-chain subunit encoded by the A and B genes, respectively.
Defects in some MHC class II genes have been associated with autoimmune disorders such as arthritis and diabetes (Otsuka et al. 2006. Proc Natl Acad Sci USA. 103: 14465-7; Aoki et al. 2005. Autoimmun Rev. 4: 373-9). Similarly, polymorphisms in MHC class II genes have been associated with susceptibility to a range of infectious diseases including malaria, tuberculosis, leprosy, typhoid fever, hepatitis and HIV/AIDS. For instance, experimental autoimmune encephalomyelitis in rhesus macaques, a model for the human disease multiple sclerosis, is known to be influenced by certain MHC class II alleles (Slierendrecht et al. 1995. International Immunology, Vol. 7: 1671-1679).
In the human population, five major -DRB region configurations are classified. These region configurations share an invariant HLA-DRA gene and a -DRB9 gene segment but differ in physical length and also in the composition and number of other DRB loci. Like humans, other primates such as chimpanzees, gorillas, and rhesus macaques have variable numbers of MHC-DRB loci per haplotype (Doxiadis et al. 2000. J. of Immunol. 164: 3193-3199). For example, the DRB region configuration in rhesus macaques (Macaca mulatta), termed Mamu-DRB, is highly plastic and has been subject to various contractions and expansions (Slierendregt et al. 1994. J Immunol 152: 2298-307). In humans a high degree of polymorphism is observed for the DRB1 locus present on all haplotypes (Robinson et al. 2003. Nucleic Acids Res 31: 311-4). In rhesus macaques, however, a high degree of region configuration polymorphism has been described characterized by marked differences with regard to number and content of distinct loci present per haplotype (Doxiadis et al. 2000. J Immunol 164: 3193-9; Doxiadis et al. 2001. Immunol Rev 183: 76-85). The Mamu-DRB region configurations themselves, however, display a relatively low degree of polymorphism. Most of the Mamu-DRB alleles belong to loci/lineages that are shared with humans (Bontrop et al. 1999. Immunol Rev 167: 339-50), and their alleles have been named accordingly (Klein et al. 1990. Immunogenetics 31: 217-9). Similar observations regarding sharing of certain MHC-DRB loci/lineages with human orthologues have been made for other primate species, whereas DRB alleles are mostly species specific (Bontrop et al. 1999. Immunol Rev 167: 339-50).
It is generally accepted that allelic polymorphisms of the MHC class II genes warrant that different allotypes select distinct peptides for T cell activation, preventing one particular pathogen from sweeping through an entire population. Most sequence variability is confined to exon 2 of the MHC-DPB, -DQA, -DQB, and -DRB genes. One of the most polymorphic regions in humans is the HLA-DRB region with more than 500 alleles described worldwide until now (Bodmer et al. 1999. Eur J. Immunogenet. 26: 81). In rhesus macaques, a species far less analysed than humans, already more than 135 Mamu-DRB distinct genes/alleles have been determined, a number which will increase rapidly when more animals are analysed.
Due to the complexity of the DRB-region, the existing typing procedures, involving single-strand conformation polymorphism, denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism analyses, or sequence analyses, are cumbersome and time consuming. It is therefore an object of the present invention to provide a typing procedure for the mammalian DRB region that allows an easy, economical, high resolution, fast and accurate haplotyping protocol.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1A: HLA-DRB exon 2 sequences subjected to phylogenetic analyses, on which D6S2878 sequences have been superimposed.
FIG. 1B: HLA-DRB exon 2 sequences subjected to phylogenetic analyses, on which D6S2878 sequences have been superimposed.
FIG. 2A: Mamu-DRB exon 2 sequences subjected to phylogenetic analyses, on which D6S2878 sequences have been superimposed.
FIG. 2B: Mamu-DRB exon 2 sequences subjected to phylogenetic analyses, on which D6S2878 sequences have been superimposed.
FIG. 3A: Organization of the human (HLA) DR region.
FIG. 3B: Partial nucleotide alignment of DRB exon 2 to intron 2. Primer sequences of different publications are indicated (Doxiadis et al., PNAS 2007, 104, 8907-8912).
FIG. 4: Organization of the DRB region of the chimpanzee (Patr-DRB).
FIG. 5: Organization of the DRB region of humans and rhesus macaques: a comparison.
FIG. 6A: Results: HLA-STR Example of genotyping of human DRB alleles (HLA) by DRB-STR microsatellite analysis.
FIG. 6B: Results: HLA-STR Example of genotyping of human DRB alleles (HLA) by DRB-STR microsatellite analysis.
FIG. 7A: Example of genotyping of DRB alleles of the chimpanzee (Patr) by DRB-STR microsatellite analysis.
FIG. 7B: Example of genotyping of DRB alleles of the chimpanzee (Patr) by DRB-STR microsatellite analysis.
FIG. 8A: Example of genotyping of DRB alleles of the rhesus macaque (Mamu) by DRB-STR microsatellite analysis.
FIG. 8B: Example of genotyping of DRB alleles of the rhesus macaque (Mamu) by DRB-STR microsatellite analysis.
FIG. 9A: Example of genotyping of DRB alleles of the cynomolgus macaque (Mafa) by DRB-STR microsatellite analysis.
FIG. 9B: Example of genotyping of DRB alleles of the cynomolgus macaque (Mafa) by DRB-STR microsatellite analysis.
FIG. 10: Example of a paternal and maternal haplotype discrimination by DRB-STR analysis in the chimpanzee (Patr).
FIG. 11: The use of DRB-STR typing for paternity testing: Example of 2 possible chimpanzee fathers.
FIG. 12: Pedigree of a cynomolgus macaque family.



DETAILED DESCRIPTION OF THE INVENTION
In one aspect the present invention provides a method for determining a DRB haplotype in a sample comprising nucleic acid from a mammalian cell, said method comprising amplifying at least an intron 2 microsatellite-containing part of at least one DRB-gene from said nucleic acid; determining the type of intron 2 microsatellite present in said at least one DRB-gene; and determining the DRB haplotype of said cell from said type of intron 2 microsatellite present in said at least one DRB-gene.
The inventors have established that the type of intron 2 microsatellite present in said at least one DRB-gene is indicative of the DRB-allele that is associated with said intron in a cis-configuration, thus allowing the identification of said DRB-allele by typing of the associated intron 2 microsatellite. An advantage of said method is that the typing of said intron 2 microsatellite can be performed by relatively simple methods which do not involve complicated methods such as single-strand conformation polymorphism or sequence analyses. Therefore, said method provides an easy and economical method for determining a DRB haplotype in a sample.
A complex dinucleotide repeat or microsatellite, D6S2878, is located in the beginning of intron 2 of the DRB genes of human and non-human primates (Bergstroem et al. 1999. Am J Hum Genet 64: 1709-1718; Epplen et al. 1997. Hum Genet 99: 399-406; Riess et al. 1990. Immunogenetics 32:110-116). This microsatellite displays a complex polymorphism profile concerning repeat length variability as well as sequence variation. According to the present invention, the variability of said microsatellite can be used for typing of the cis-associated DRB-allele. The number of and identity of distinct DRB-alleles that are present in a sample, as determined by the number and types of intron 2 microsatellites that are present in said sample, is used to determine a DRB-haplotype.
The number and types of intron 2 microsatellites that are present in a sample can be determined by any method known in the art, including but not limited to sequence analyses and sequence-specific amplification. However, a quick and convenient method for determining the number and types of intron 2 microsatellites comprises the determination of the length of the microsatellite, as it was found that the length of the intron 2 microsatellite is indicative for the cis-associated DRB-allele.
Therefore, in a preferred embodiment the invention provides a method for determining a DRB haplotype, wherein the typing of the intron 2 microsatellite is based on the length of said microsatellite.
Thus, a DRB-haplotype can be determined by analysis of the length of intron 2 microsatellite that is present in the amplified fragments from the at least one DRB gene. Each haplotype is identified by a specific number and size of the amplified fragments. Thus by identifying the specific number and size of the amplified fragments, the haplotype can be identified.
The number of distinct fragments obtained after amplification will depend on the number of distinct DRB-alleles that are present in a particular DRB-haplotype. The total number of DRB-alleles that can be present in a sample, and consequently resulting in multiple distinct amplified fragments, also depends on whether the cell is homozygous or heterozygous for a DRB-haplotype, as in the latter case more distinct fragments might be present.
In a preferred embodiment, the type of DRB-allele is determined by comparing the amplified intron 2 microsatellite-derived fragments with a reference. Said reference can be a sample from a individual of which the types of DRB-alleles and the corresponding DRB-haplotype have been previously determined. Said reference can, for example, be taken along in the amplification reaction. The reference sample can be taken from the same or a different species, however, it is preferred that said reference sample is taken from the same species.
Therefore, the invention also provides a method wherein a DRB haplotype is determined by comparing the detected types of intron 2 microsatellite with a reference.
In preferred embodiment, said reference is taken from a sample from an individual of which the types of DRB-genes and the corresponding DRB-haplotype have been previously determined and of which relevant data, comprising number and length and/or sequence of the amplified intron 2 microsatellite fragments, have been stored in a database.
Therefore, in this embodiment, the invention provides a method wherein a type of intron 2 microsatellite is determined by comparing with a reference comprising a database of DRB haplotypes correlated with the associated intron 2 microsatellite types.
In a preferred embodiment, a database according to the invention is present in an electronic storage device, such as, but not limited to, a computer or a server. It is further preferred that said database comprising said reference can be addressed to compare the amplified intron 2 microsatellite-derived fragments with said reference.
Amplification of at least intron 2, or a microsatellite-containing portion thereof, of a DRB-gene can be performed by any method known in the art including, but not limited to, polymerase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, rolling circle amplification technology, and transcription-mediated amplification. Each of these amplification methods uses different approaches to achieve the amplification of nucleic acid molecules to amounts that can subsequently be detected.
In a preferred embodiment, the invention also provides a primer pair that spans the region containing said intron 2 microsatellite, for amplifying at least said intron 2 microsatellite-containing part of at least one DRB-gene.
According to this embodiment, said primer pair is preferably selected to allow amplification of at least 1 DRB gene, more preferred at least 2 DRB genes, more preferred at least 3 DRB genes, more preferred at least 4 DRB genes, more preferred at least 5 DRB genes, more preferred at least 6 DRB genes, more preferred at least 7 DRB genes, more preferred at least 10 DRB genes, that are present within the DRB region of one or more individuals of a species.
Therefore, each of the primers of said primer pair preferably is selected to hybridize to a nucleic acid region that is conserved in most or essentially all DRB-genes and alleles thereof within a species, thereby allowing the amplification of said intron 2 microsatellite of essentially all DRB genes and alleles thereof that are present within the DRB region of one or more individuals of a species.
In a preferred embodiment, said primer pair comprises a first primer of which the nucleotide sequences corresponds to a conserved nucleotide sequence that is present in a position that is adjacent to the microsatellite in intron 2, and a second primer of which the nucleotide sequences corresponds to a conserved sequence that is present on the opposite side of said microsatellite sequence, relative to the first primer.
In a preferred embodiment, said first primer is selected from a region spanning the 3′ end of exon 2 and the 5′start of intron 2 of a DRB gene. This region was found to be conserved within species, but not between species. A second primer can be selected from a region comprising a conserved sequence, said region being present within a range of up to 1000 nucleotides from the microsatellite at the opposite side of the microsatellite in intron 2 of a DRB gene relative to said first primer.
In a more preferred embodiment, said second primer is selected from a region comprising conserved sequences that are present within a range of 5 to 60 nucleotides from the microsatellite at the opposite side of the microsatellite in intron 2 of a DRB gene, relative to said one primer.
Therefore, in an even further preferred embodiment, a primer pair according to the invention comprises a first primer of which the nucleotide sequences corresponds to a conserved region spanning the 3′ end of exon 2 and the 5′ start of intron 2 of a DRB gene, and a second primer of which the nucleotide sequences corresponds to a region comprising conserved sequences that is present within a range of 5 to 60 nucleotides from the microsatellite at the opposite side of the microsatellite in intron 2 of a DRB gene, relative to said first primer.
In yet an even further preferred embodiment, said first primer is chosen from the primers









(SEQ ID NO 1)



GAGAGCTTCACAGTGCAGC;



 



(SEQ ID NO 2)



TTCACAGTGCAGCGGCGAGGT;



and



 



(SEQ ID NO 6)



CGTGTCCCCACAGCACGTTTC 






and said second primer is chosen from the primers










(SEQ ID NO 3)



GAGAGGATTCTAAATGCTCAC;



 



(SEQ ID NO 4)



ACACCTGTGCCCTCAGAACT;



and



 



(SEQ ID NO 5)



ACATCTGTGTCCTCAGACCT.






It is furthermore preferred that the amplified nucleic acid molecules comprising at least intron 2 of a DRB gene, or a microsatellite-containing portion thereof that are derived from different DRB-alleles can be discriminated by physical characteristics such as size, isoelectric point, or nucleotide sequence of the molecules. Therefore, the length of the amplified fragment is preferably less than 1000 nucleotides, more preferred less than 900 nucleotides, more preferred less than 800 nucleotides, more preferred less than 700 nucleotides, more preferred less than 600 nucleotides, more preferred less than 500 nucleotides, more preferred less than 400 nucleotides, more preferred less than 300 nucleotides.
An amplified fragment comprising the microsatellite can be detected by any method known in the art. Detection methods include real-time detection methods such as DNA binding fluorophores, 5′ endonuclease, adjacent linear and hairpin oligoprobes, and self-fluorescing amplicons.
In another embodiment, detection of the amplified nucleic acid is performed by post-amplification methods, including but not limited to, colorimetric detection, chemiluminescence, and gel electrophoresis detection.
Gel electrophoresis comprises the analysis of nucleic acid molecules by using a polymer such as agarose or polyacrylamide. Known methods include slab gel electrophoresis and capillary gel electrophoresis. Nucleic acid molecules are separated on the polymer based on physical characteristics such as size, shape, or isoelectric point, of the molecules. Visualization of nucleic acid molecules after electrophoreses comprises classic staining procedures, including but not limited to ethidium bromide, silver, SYBR Green (2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium; Invitrogen), coomassie blue dyes, or inorganic microparticle labels such as nanocrystals or quantum dots.
The length of an amplified nucleic acid can be determined by methods known to a skilled person, including but not limited to comparison to a known standard provided by fragments of known length, measuring of the time that is required to reach a defined position on the gel under standard conditions, and by any other method that determines the size, shape, or isoelectric point of the nucleic acid, such as mass spectrometry.
Sequence analyses of each of the amplified nucleic acid molecules, or of a suitable part thereof, can be performed using methods that are known in the art, including but not limited to, chemical analyses, enzymatic analyses using nucleotide analogues, and hybridization methods.
In a preferred embodiment, amplified nucleic acid molecules according to the invention are generated using labelled nucleotide analogues in the amplification reaction, allowing detection of said amplified nucleic acid molecules after gel electrophoresis without staining and washing procedures. Labelled nucleotide analogues that can be used include, but are not limited to, fluorescently-labelled nucleotide analogues such as cyanine-dyes (Cy3 or Cy5)-labelled analogues, and radioactively labelled nucleotide analogues.
In an alternative embodiment, amplified nucleic acid molecules are detected through the native fluorescence of nucleic acid molecules.
In a more preferred embodiment, the nucleic acid molecules are fluorescently labelled. They can be detected using any method known in the art, such as, for example, laser-induced fluorescence. Detection is based on the excitation and emission spectra of the fluorescent label that is used.
In an even more preferred embodiment, the amplified nucleic acid molecules are generated using at least one primer that is labelled. Said at least one primer can be labelled with, for example, luminescent method system such as lanthanide ions, or fluorescence-based dyes such as 6-FAM, HEX, NED, ROX, 5-FAM, JOE, Cy3, and Cy5. The use of at least one primer that is labelled will also allow detection of the amplified nucleic acid molecules by gel electrophoresis without staining and washing procedures.
Therefore, a preferred method according to the invention comprises amplifying at least an intron 2 microsatellite-containing part from a DRB gene with a primer pair, wherein one or more of the primers is labelled.
Said sample comprising nucleic acid molecules of the cell that is used for amplifying at least intron 2, or a microsatellite-containing portion thereof, may comprise any nucleic acid molecule that comprises intron 2, or at least a microsatellite-containing portion thereof. These nucleic acid molecules include, but are not limited to, unspliced RNA, such as, for example, nuclear RNA, and genomic DNA.
In a preferred embodiment, the invention provides a method for determining a DRB haplotype in a sample comprising genomic DNA.
The sample can be derived from any mammal that comprises a microsatellite in intron 2 of a DRB gene. The present invention is suitable for designing breeding programs such as used in animal husbandry and for endangered species such as whales and elephants. Non-limiting examples of a mammal comprise pets such as dog and cat; ungulates including sheep, goat, and cattle such as cow; horse; and primates.
In a preferred embodiment, the sample is derived from any human or non-human primate that comprises a microsatellite in intron 2 of a DRB gene. Not limiting examples of human or non-human primates include apes such as chimpanzee, gorilla, gibbon, siamang, orangutan, and human, Old World monkeys such as mandrill, macaque and baboon, New World monkeys, and Prosimians.
In a further preferred embodiment, a method according to the invention provides determining a DRB-haplotype in a sample that is derived from a human.
In another aspect, the invention relates to a kit for determining a DRB haplotype in a cell, said kit comprising means for amplifying at least an intron 2 microsatellite-containing part of at least one DRB-gene.
In one embodiment, said kit comprises a pair of primers that span a region of a DRB gene flanking the intron 2 microsatellite-containing part of at least one DRB-gene.
In a preferred embodiment, a first primer of said primer pair comprises sequences selected from a conserved region spanning the 3′ end of exon 2 and the 5′ start of intron 2 of a DRB gene, while a second primer comprises sequences selected from a conserved region that is present on the opposite side of said microsatellite sequence, relative to the first primer.
In a further preferred embodiment, said first and second primers are selected such that the length of the amplified fragment from the intron-2 of a DRB-gene is less than 1000 nucleotides, more preferred less than 900 nucleotides, more preferred less than 800 nucleotides, more preferred less than 700 nucleotides, more preferred less than 600 nucleotides, more preferred less than 500 nucleotides, more preferred less than 400 nucleotides, more preferred less than 300 nucleotides.
In another embodiment, the kit comprises primers of which a 5′ primer comprises the nucleotide sequence of SEQ ID NO 1; and a 3′ primer comprises the nucleotide sequence of SEQ ID NO 3.
In yet another embodiment, the kit comprises primers of which a 5′ primer comprises the nucleotide sequence of SEQ ID NO 2; and a 3′ primer comprises the nucleotide sequence of SEQ ID NO 4 or SEQ ID NO 5.
In a further embodiment, the kit comprises primers of which a 5′ primer comprises the nucleotide sequence of SEQ ID NO 6; and a 3′ primer comprises the nucleotide sequence of SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5.
In another aspect, the invention relates to the use of a method for determining a DRB haplotype in a sample for a genetic application.
The genes of the MHC are known to be among the most polymorphic loci known in human or non-human primates, due to a high rate of mutation and recombination. Major differences between species reside not only in the number and identity of MHC genes, but also in the degree of allelic polymorphism for individual genes. Therefore, the method for determining a DRB-haplotype in a sample, as provided by the current invention, can be used in genetic tests. Genetic test comprise tests to look for a possible predisposition to disease before any symptoms appear.
The most widespread type of genetic testing is newborn screening. Genetic test can be helpful in several areas: early detection, diagnosis, prognosis, and treatment.
In a further embodiment, the method for determining a DRB-haplotype in a sample according to the present invention can be used a genetic application comprising paternity testing.
Paternity tests can be performed postnatally by, for example, testing of blood or testing of a buccal swab, or testing of the umbilical cord, or testing of other sample collection including but not limited to semen, tissue and hair. Paternity tests can also be performed pre-natally by testing of a sample obtained by amniocentesis or by chorionic villus sampling.
In yet a further embodiment, the method for determining a DRB-haplotype in a sample according to the present invention can be used in a genetic application comprising forensic testing.
Forensic testing includes the application of method for comparison of biological samples in criminal investigations. Typically, the samples are processed in a dedicated forensic laboratory. Short tandem repeats such as provided by the microsatellite in the intron 2 of a DRB-gene may be included in a standard battery of core loci. This may increase the discriminatory power and decrease the probability of a match between profiles of two unrelated persons.
In another embodiment, the method for determining a DRB-haplotype in a sample according to the present invention can be used in a genetic application comprising tissue typing for transplantation testing.
MHC molecules play a crucial role in T-cell activation by antigen presenting cells. Antigenic recognition depends on the interaction between the antigenic peptide-binding MHC molecule and the T-Cell Receptor (TCR), an immunoglobulin-like heterodimeric protein expressed on T-lymphocytes. The transplanted organ represents a continuous source of antigens that can induce a rejection response at any time post-transplant. Furthermore, donor-derived immuno-competent lymphocytes may react with MHC-incompatible recipient cells and induce inflammatory responses in host tissues such as the skin and gastrointestinal tract. This complication is frequent after bone marrow transplantation, but may also affect recipients of liver and other organ transplants and even blood transfusions.
Therefore, transplants with well-matched antigens, especially matching of DRB-haplotype, may function significantly better than those with a poor match, due to different rates of initial immune reactions, graft rejection, and graft failure due to infection or other causes.
In yet another embodiment, the method for determining a DRB-haplotype in a sample according to the present invention can be used in a genetic application comprising testing for chronic or infectious diseases.
Determining a DRB-haplotype may help to determine a possible predisposition to a disease before any symptoms appear. These diseases include, but are not limited to, autoimmune disorders such as arthritis, diabetes, multiple sclerosis (Gregersen et al., 2006. Nature 443: 574-7), bowel diseases such as ulcerative colitis and Crohn's disease, psoriasis, and chronic fatigue syndrome. Furthermore, polymorphisms in MHC class II genes such as DRB genes, have been associated with susceptibility to a range of infectious diseases including malaria, tuberculosis, leprosy, typhoid fever, hepatitis and HIV/AIDS.
In yet another aspect, the invention relates to the use of a kit for determining a DRB haplotype in a cell, said kit comprising means for amplifying at least an intron 2 microsatellite-containing part of at least one DRB-gene, in a genetic application comprising paternity testing, forensic testing, tissue typing for transplantation testing, or testing for chronic or infectious diseases.
The DR region of humans (HLA-DR) as well as other primates as chimpanzees and macaques and other mammals as sheep consist of one DRA and one to several DRB loci on each chromosome (here called ‘DRB region configurations’). These DRB region configurations vary in number and content of DRB loci present. Examples are given for humans (FIG. 3), chimpanzees (FIG. 4), and rhesus macaques in comparison to humans (FIG. 5). Certain DRB loci are highly polymorphic. Thus, DRB region configurations may display allelic polymorphism and are then called ‘DRB haplotypes’. In humans only 5 DRB region configurations are known that are highly polymorphic especially at the DRB1 locus, whereas in rhesus macaques more than 30 region configurations are described which display a low degree of polymorphism (FIG. 5).
The present invention describes the possibility to amplify the STR present in intron 2 of all DRB loci, not only DRB1, that contain an exon 2/intron 2 sequence thus all except DRB2 and DRB8. The result of this high-throughput, high-resolution microsatellite typing is a DRB-STR pattern that specifies a given DRB haplotype. Examples for such a DRB-STR length analysis are given for humans (FIG. 6), chimpanzees (FIG. 7), rhesus macaques (FIG. 8) and cynomolgus macaques (FIG. 9). The resulting DRB haplotypes are color-coded. Extensive DRB-STR length analyses of Mhc homozygous individuals or of pedigreed animals of more than 3 generations resulted in the definition of DRB haplotypes of humans and rhesus macaques. Additionally, definition of DRB haplotypes was feasable for chimpanzees (Patr-DRB, Table 4) and cynomalogus macaques (Mafa-DRB, Table 5). Comparison of FIG. 7 and Table 4 shows that the present application allows a definition of 13 instead of six DRB region configurations in the chimpanzee.
The present invention describes the possibility of high resolution typing via DRB haplotyping, not DR/DQ haplotype association studies as claimed by U.S. Pat. No. 5,908,749 and US 2003/108940 A1. The present invention allows DRB typing in even a higher resolution than exon 2 sequencing, the method-of-choice used for high-resolution typing (van Dijk et al. 2007). An example is given in FIG. 10 for a pedigreed chimpanzee family in which the differentiation between haplotype b of father (Frits) and haplotype d of mother (Sonja) was only possible by DRB-STR typing. Thus, DRB-STR typing as described in the present invention is a powerful method for paternity testing (examples given in FIGS. 10 and 11). Additionally, the present invention allows powerful and quick parentage analysis as needed for example in macaque colony management (FIG. 12).
Furthermore, DRB high resolution typing is useful for disease association studies. In humans it is known that genes of the Major Histocompatibility Complex (Mhc) encode proteins important for activating antigen-specific immune responses. Alleles of the human Mhc class I as well as class II genes are known to be associated with susceptibility as well as resistance to certain autoimmune diseases. Alleles of the HLA-DRB loci are described to be tightly linked with various immune-related diseases as sarcoidosis (Grosser et al. 2005) and chronic pancreatitis (Cavestro et al. 2003). Especially for autoimmune diseases as Addisons' disease, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), narcolepsia, multiple sclerosis (MS), and type 1 diabetes (T1D) (Carrington 1999; Shiina et al. 2004) (summarized in Shiina, T A, 2006 and reviewed by Carrington 1999) specific DRB alleles or DR/DQ allele combinations are known to be associated with disease susceptibility and/or protection. In RA and Addisons' disease, specific DRB1*04 alleles have been reported to be linked to disease susceptibility (Fries et al. 2002; Gombos et al. 2007). Thus, a high resolution typing for DRB is necessary for the screening of patients for both disorders. Numerous studies have shown a high linkage disequilibrium with HLA-DRB1*1501 with susceptibility to SLE (Graham et al. 2007), Graves' disease (Sasaki et al. 2007), narcolepsy (Planelles et al. 1997) as well as to multiple sclerosis (MS). In narcolepsy and MS disease susceptibility has been fine mapped to the HLA class II haplotype including the HLA-DRB5*0101 allele (Dyment et al. 2004; Dyment et al. 2005; Fogdell et al. 1995; Ramagopalan et al. 2007). The DRB-STR typing method presented here allows high resolution typing and therefore can discriminate between the susceptible alleles DRB1*1501 and DRB5*0101 and their variants DRB1*1502 and DRB5*0201, respectively. This discrimination is otherwise only possible by time-consuming and expensive sequencing techniques. Furthermore, both DRB alleles are shown to encode DR beta chains, which form two different HLA-DR dimers that are both, expressed at significant levels on the cell surface (Prat et al. 2005). Additionally, recent publications showed that epistatic interactions of the two DR molecules have a major influence on the disease development in MS patients (Gregersen et al. 2006). Thus, typing only for the DRB1 allele that is mostly performed by DRB typing procedures may not be sufficient for MS research in the future. On contrast, several chromosomal regions have been linked to T1D susceptibility in humans using modern genome screening methods. The largest contribution comes, however, from several genes in the Mhc complex, formerly known as DR3/DR4. There is evidence that certain residues important for structure and function of both HLA-DQ and -DR peptide binding pockets determine disease susceptibility and resistance (Pociot and McDermott 2002; Zavattari et al. 2001). A number of susceptible, neutral, and protective DR-DQ haplotypes have been identified recently by the T1D Genetics Consortium (Erlich et al. 2008). The most susceptible haplotypes involve DRB1*0301 and DRB1*0405/0401/0402, whereas the most protective ones are DRB1*1501 and DRB1*1401. However, allelic variation of DRB can be observed for disease association in different ethnic populations (Al-Harbi et al. 2004; Almawi et al. 2004). Thus, for screening of T1D patients as well as for further fine mapping analysis of other, probably disease associated, loci as DRB5, a quick typing method as described here will be needed.
Definitions
The term DRB-gene is defined as a functional unit whose inheritance can be followed experimentally. The term DRB gene comprises both an active gene and a pseudogene, the latter being defined as a sequence that closely resembles a known DRB-gene but which is not expressed.
The term DRB-allele is defined as an alternative form of a DRB-gene that can be distinguished from other forms of said DRB-gene by, for example, sequence analyses.
The term DRB-locus is defined as a position on a chromosome that corresponds to a DRB-gene.
The term DRB-haplotype is defined as the unique combination of one or more DRB genes or their evolutionary equivalents that are in most mammals associated in a cis-configuration to the MHC-DRA gene or its evolutionary equivalent on the same chromosome. A DRB-haplotype is determined by the number of DRB-genes, and the specific DRB-alleles that are present of each of the DRB-genes, within the DRB region.
EXAMPLES
Example 1
Materials and Methods
Samples
The 167 rhesus monkeys analyzed, housed at BPRC's breeding colony, originated mostly from India, but some are also of Burmese or Chinese origin. Seven of these animals are completely homozygous for their MHC region and derived from consanguineous matings; two additional animals are homozygous for their Mamu-A, -B, and -DR serotypes. Genomic DNA of human individuals or rhesus macaques was extracted from EDTA blood samples or from immortalized B-cell lines using a standard salting out procedure. Of the 160 human samples tested, 64 were HLA-DRB homozygous, 17 of which belong to a thoroughly characterized homozygous typing cell panel of the XIV International Histocompatibility Workshop, 2005.
STR-DRB Genotyping
The relevant DNA segment in rhesus macaques was amplified with a forward primer located at the end of exon 2 (5′Mamu-DRB-STR: TTC ACA GTG CAG CGG CGA GGT) (SEQ ID NO: 2) and 2 labeled reverse primers in intron 2 (3′Mamu-DRB-STR_VIC: ACA CCT GTG CCC TCA GAA CT (SEQ ID NO: 4) and 3′Mamu-DRB-STR_FAM_1007: ACA TCT GTG TCC TCA GAC CT (SEQ ID NO: 5)). For human samples a labeled forward primer located at the end of exon 2 (5′HLA-DRB-STR_VIC: GAG AGC TTC ACA GTG CAG C) (SEQ ID NO: 1) and one reverse primer in intron 2 (3′HLA-DRB-STR: GAG AGG ATT CTA AAT GCT CAC) (SEQ ID NO: 3) were used. The labeled primers were synthesized by Applied Biosystems (Foster City, USA) and the unlabeled primers by Invitrogen (Paisley, Scotland). The PCR reaction for rhesus macaques was performed in a 25 ml reaction volume containing 1 unit of Taq polymerase (Invitrogen, Paisley, Scotland) with 0.6 mM of the unlabeled forward primer, 0.4 mM of the VIC labeled reverse primer, 0.2 mM of the FAM labeled reverse primer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 1×PCR buffer II (Invitrogen, Paisley, Scotland) and 100 ng DNA.
The PCR mixture for the human STR amplification was the same as that used for rhesus macaques with 0.1 mM of the VIC labeled forward primer and 0.1 mM of the unlabeled reverse primer. The cycling parameters for both amplifications were a 5 mM 94° C. initial denaturation step, followed by 5 cycles of 1 mM at 94° C., 45 s at 58° C., and 45 s at 72° C. Then the program was followed by 25 cycles of 45 s at 94° C., 30 s at 58° C. and 45 s at 72° C. A final extension step was performed at 72° C. for 30 mM. The amplified DNA was prepared for genotyping according to the manufacturer's guidelines and analyzed on an ABI 3130 genetic analyzer (Applied Biosystems). STR, analysis was performed with the Genemapper program (Applied Biosystems) and all samples were analyzed at least twice.
PCR, Cloning and Sequencing
Seventy-five different Mamu-DRB alleles and 38 HLA-DRB alleles were sequenced from exon 2 to intron 2 including the microsatellite with a generic 5′ DRB-exon 2 primer CGT GTC CCC ACA GCA CGT TTC (SEQ ID NO: 6) together with the same 3′ primers as used for DRB-STR genotyping but without label. The PCR reactions for rhesus monkey and human DRB were performed in a 100 ml volume containing 4 units of Taq polymerase (Invitrogen, Paisley, Scotland) with 0.2 mM of each primer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 1×PCR buffer II (Invitrogen, Paisley, Scotland) and 200 ng DNA. The cycling parameters were the same as described for STR-DRB genotyping. The resulting amplicons were cloned and sequenced as described recently (Doxiadis et al. 2006. Immunogenetics 58: 259-268; Penedo et al. 2005. Immunogenetics 57, 198-209). The resulting sequences were analyzed using the Sequence Navigator program (Applied Biosystems).
Phylogenetic Analyses
Multiple sequence alignments of exon 2 of human and rhesus macaque -DRB sequences were created using MacVectorTM version 8.1.1 (Oxford Molecular Group) and phylogenetic analyses was then performed using PAUP version 4.0b.10 (Swafford 2002. PAUP*.Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4 (Sinauer Associates, Sunderland, Mass.)). Pairwise distances were calculated using Kimura-2 parameter and the neighbor-joining method for creating a phylogram. Confidence estimates of grouping were calculated according to the bootstrap method generated from 1,000 replicates.
Results
Mamu-DRB Region Configuration Definition by Microsatellite Markers
The rhesus macaques studied are part of a large self-sustaining colony that has been thoroughly pedigreed based on segregation of serologically and molecularly defined markers (Doxiadis et al. 2001. Immunol Rev 183: 76-85; de Groot et al. 2004. J Immunol 172: 6152-7). The current panel covers 22 different region configurations characterized by unique combinations from two to six distinct Mamu-DRB genes (Table 1). Limited allelic polymorphism is detected within region configurations #1, 11, 15, 18, 19, and 21. To denote allelic variation observed, for instance, in region configuration #1, the relevant haplotypes have been designated 1a and 1b (Table 1). The gene frequencies of the different region configurations as encountered in our population of animals are provided as well, in concert with the number of animals tested. Some haplotypes are frequently observed whereas others appear to be rare (Table 3).
Nearly all Mamu-DRB loci/lineages possess the D6S2878 microsatellite, and the relevant DNA segment could be amplified by means of the unique primer set developed for this protocol (Table 1). Amplification failures have only been observed for a few DRB6/DRBW pseudogenes for which the corresponding amplicons could be scarcely detected, most probably due to primer inconsistencies. The overall results illustrate, however, that the lengths of the amplified STR products are highly variable and range from 153 to 293 bp (Table 1). Additionally, various STR markers seem to be predictive for the presence of a particular Mamu-DRB allele and, moreover, family studies demonstrated that they segregate in a Mendelian manner. Subsequent sequencing of the DNA segment ranging from exon 2 to intron 2 was conducted to unequivocally link each D6S2878 allele to an individual DRB gene/allele. This approach proved that diverse STR lengths could distinguish even highly similar alleles, differing for only one or two nucleotides. An instance is provided by the Mamu-DRB1*07032 and 07033 alleles, which are part of the two haplotypes belonging to configuration #18 (Table 1).
On average, most of the STRs linked to an individual allele appear to be rather conservative in composition and length. In the case of the frequently observed haplotype 11a (Table 3), for example, no length variability for both DRB1 gene-associated D6S2878 alleles is observed, which also holds true for the STR that is linked to DRB5*0301 as seen in haplotype 1a (Table 1). The DRB1*0406-linked STR of the latter configuration, however, may slightly vary in length. Such differences, as can be seen for example in haplotype 12, are reproducible, and do segregate in families with a particular haplotype as defined by serological methods (Bontrop et al. 1995. Immunol Rev 143: 33-62). As each region configuration is composed of an exclusive combination of different Mamu-DRB genes, the most essential conclusion that can be drawn is that the combination of STR markers appears to be unique for a given region configuration/haplotype.
HLA-DRB Haplotype Definition by Microsatellite Analysis
Genotyping for the highly divergent (GT)x(GA)y microsatellite allows speedy and accurate DR haplotyping in rhesus macaques, a species known to possess a high number of region configurations; these display, however, low levels of allelic variation. Next, it was investigated whether the same approach would work in a species, that possesses a relatively low number of region configurations that parade abundant levels of allelic polymorphism. Therefore, in the first instance, a thoroughly characterized panel of 160 unrelated human samples of Caucasoid origin was chosen to conduct such an analysis. This selection also comprised 64 homozygous typing cells, facilitating the simple definition of haplotypes. Furthermore, the panel included the five known HLA-DRB region configurations, designated DR8, DR1, DR51, DR52, and DR53 (Schreuder et al. 2005. Tissue Antigens 65: 1-55), but also covered the 13 most common DR serotypes present in the Caucasoid population, differing for their DRB1 alleles (Table 2). To denote allelic variation as observed within some serotypes, the different DRB haplotypes have been designated a, b, c and so on (Table 2). The number of samples tested per haplotype, as well as the gene frequencies of the serotypes (Marsh et al. 2000. The HLA FactsBook (Academic Press, London, UK, San Diego)), have also been summarized (Table 3).
Again, a specially designed generic primer pair allowed amplification of the relevant intron 2 segment for virtually all HLA-DRB genes/alleles. Amplification failed only for one particular HLA-DRB5 allele present on DR16 haplotypes. This may be due to a mutated primer site. Subsequent extensive sequencing of exon 2-intron 2 DNA segments verified for each of the HLA-DRB alleles the unique linkage to its adjacent D6S2878 allele. The lengths of the respective repeats are highly polymorphic in the human population as well and range from 135 to 220 bp (Table 2). Moreover, STR lengths are highly predictive for the presence of individual HLA-DRB alleles, as was also shown earlier for rhesus monkeys. Again, differential STR lengths can make the distinction between highly similar -DRB alleles, differing for a few nucleotides: for example in the case of DRB1*0801(03) and DRB1*080302 (Table 2).
The DR53 region configuration contains a pseudogene, named HLA-DRB7, which is absent in macaques. This pseudogene displays no allelic variation, and indeed the associated D6S2878 is invariant in length independent of the adjacent -DRB1 allele. Thus, the DRB7-associated STR typifies all the haplotypes belonging to the DR53 region configuration (Table 2). The same holds true for the DRB6 allele and its adjacent STR, which are characteristic for the DR1 region configuration, covering the DR1 and DR10 serotypes. As observed in rhesus macaques, some HLA-DRB alleles appear to be associated with multiple D6S2878 variants. A case is provided by the HLA-DRB1*140101 allele observed within the DR14 haplotypes, but some of the repeats grouping in the DR11a and DR13c family of haplotypes also show length differences (Table 2). Family analyses are needed to demonstrate their segregation. Most of the human repeats, however, appear to be conservative in composition and length, and are linked to an individual allele. Within this panel of 30 different HLA-DRB haplotypes, all but two can be readily dissected based on by their D6S2878 profiles. Thus, complex DR region configuration/haplotype information, as present in at least two populations of primate species, is readily obtained and defined based on D6S2878 genotyping after a simple amplification protocol conducted with one primer set.
Genetic Stability and Evolutionary History of the STR-DRB Complex
The D6S2878 microsatellite has a composite character in primates (Riess et al. 1990. Immunogenetics 32: 110-6; Bergstrom et al. 1999. Am J Hum Genet 64: 1709-18; Kriener et al. 2000. Immunogenetics 51: 169-78; Trtkova et al. 1995. Mol Phylogenet Evol 4: 408-19) and phylogenetic comparisons of different DRB1 sequences obtained from humans and chimpanzees indicated that the ancestral structure most likely must have been a (GT)x(GA)y dinucleotide repeat. The HLA-DRB1 associated microsatellite comprises three sections exhibiting different evolutionary stabilities. The 5′(GT)x repeat represents the longest segment and evolves most rapidly, which is a known feature for long, uninterrupted dinucleotide repeats. The middle section or the (GA)z part is shorter and interrupted; its constellation appears to correlate well with different lineages/loci and its length seems to segregate with specific DRB1 alleles. The length of the 3′(GA)y part appears to be specific for a certain DRB lineage/locus (Bergstrom et al. 1999. Am J Hum Genet 64: 1709-18). All HLA-DRB exon 2 sequences described in this study have been subjected to phylogenetic analyses, on which the D6S2878 sequences have been superimposed (FIG. 1). As can be seen, this report extends the knowledge on this particular microsatellite but also underscores its compound character (Epplen et al. 1997. Hum Genet 99: 399-406; Bergstrom et al. 1999. Am J Hum Genet 64: 1709-18). Additionally, a short dinucleotide (GC)1-3 part could be observed at the 3′end of the microsatellite of all HLA-DRB genes except for those belonging to the DRB6 and DRB7 pseudogenes. The variation seen in this newly recognized section of the microsatellite seems to be prognostic for certain HLA-DRB lineages or loci, respectively. The 5′(GT)x part is indeed the most polymorphic, which may especially in the case of fairly long (GT)x repeats evolve faster than the mutation rate operative on exon 2 itself (FIG. 1). This phenomenon, already described for DQCAR (Lin et al. 1998. Tissue Antigens 52: 9-18) could explain why, for instance, the HLA-DRB1*1302 allele is associated with a STR displaying length variation (Table 2).
The ancient HLA-DRB6 and -7 pseudogenes appear to miss the middle, interrupted (GA)z or the (GA)y part, respectively. The HLA-DRB6*0101 gene harbors a genetically stable D6S2878 showing no length differences at all. This is most probably due to its composition represented by a short and interrupted 5′(GT) and 3′(GA) part (Petes et al. 1997. Genetics 146: 491-8; Wierdl et al. 1997. Genetics 146: 769-79). For HLA-DRB6*0201 the opposite is true, and the long and uninterrupted (GT)x and (GA)y parts are indicative for unstable STR lengths (Table 2 and FIG. 1) (Jin et al. 1996. Proc Natl Acad Sci USA 93: 15285-8).
The Mamu-DRB exon 2 sequences have also been subjected to phylogenetic analyses and the genetic composition of the D6S2878 sequences has been superimposed (FIG. 2). Like in humans, D6S2878 has a compound character. The newly described fourth 3′(GC) dinucleotide part is also present in rhesus macaques with repeat lengths ranging from 1 to 5. Comparable to the human situation, Mamu-DRB6 alleles form a distinct Glade in the phylogenetic tree. Since the DRB6 locus is thought to predate the divergence of Old World monkeys, great apes, and homonoids, it is not surprising that the same ancestral (GT)x(GA)y structure was detected in rhesus monkeys just as in humans. In contrast to the human microsatellite, some Mamu-DRB lineages/loci are characterized by a multipart (GT)x and/or a (GA)z middle segment. In general, the rhesus macaque STR appears to be even more complex than its human equivalent. This multifaceted composition seems to correlate with the high number of DRB region configuration/haplotype diversity observed in rhesus macaques. Despite the complex microsatellite patterns observed in the rhesus macaque, the composition and length of repeats associated with known Mamu-DRB alleles, are as expected, highly similar. The rhesus macaque DRB region has been subject to several rounds of duplication and contraction processes. For that reason, it is difficult to understand which highly related genes located on different region configurations/haplotypes represent separate loci or whether these sequences have an allelic affiliation. This microsatelite will be helpful in sorting out such genetic relationships.
Discussion
Microsatellite D6S2878 is considered to represent a promising marker for the development of a quick and accurate DRB haplotyping protocol in primate species, on the condition that one single set of informative primers can be developed. Indeed, a specifically designed primer pair allowed amplification of the relevant intron 2 segment for virtually all HLA- as well as Mamu-DRB genes/alleles. For both species, amplification artifacts have rarely been observed and such types of problems are easily to overcome by designation of specific primers that can be added to the same reaction mixture. The D6S2878 STR was proven to be highly variable in length, not only in humans but also in the rhesus macaque, thus verifying this microsatellite as a useful marker for DRB typing of both species. Phylogenetic analyses of human as well as rhesus macaque exon 2 sequences have been performed and compared with microsatellite composition. For humans, the evolutionary relationships of exon 2 and the adjacent microsatellite seem to segregate closely together (FIG. 1). In rhesus macaques, the microsatellite composition was far more variable than in humans and a comparison of microsatellite and exon 2 phylogeny does not always seem to match the microsatellie composition (FIG. 2). One explanation for these results may be that rhesus macaque DRB loci/lineages are much older than their human equivalents, and this can be the reason for the higher diversity of the adjacent microsatellite as well. Furthermore, preliminary results of intron sequences illustrate that some of the Mamu-DRB sequences that are considered alleles of a given locus probably represent monomorphic loci themselves. However, truly allelic variants manifest the reliability of the comparison of exon 2 and the D6S2878 marker not only in humans but also in rhesus macaques.
Microsatellite typing was, in the first instance performed on large human and rhesus macaque panels for which the typing information was known. The most essential conclusion to be drawn (Table 1 and 2) is that in both species the combination of STR markers appears to be unique for a given haplotype. Within the rhesus macaque panel of 31 haplotypes all could be defined unambiguously, within the human panel of 30 haplotypes all but two could be thus defined. As stated earlier, ambiguities can easily be solved by development of additional primer pairs. As a control, a blind test was performed with 47 human and 26 rhesus monkey samples. More than 90% of the samples were scored correctly for their Mhc-DRB haplotypes. Some of the samples were not scored properly because they contained allotypes that were not present in the original test panel. Thus, this D6S2878 typing protocol provides a highly reliable method for Mamu- and HLA-DRB haplotyping, with its main advantage being simplicity and speed. Since differently labeled primers can be used for microsatellite typing, multiplexing is possible and 96 samples or even more can be analyzed within one test panel. The simplicity of the test is especially useful for Mamu-DRB haplotyping, which is otherwise extremely time consuming due to the unprecedented high number of different -DRB region configurations. For the human situation, this approach is very helpful in the analysis of large amounts of samples, as they are needed, for example, in population and/or disease association studies. Furthermore, this method may also be of use in forensic medicine as well as in paternity-testing protocols. Additionally, high-resolution -DRB haplotyping will simplify donor-recipient matching in organ as well as bone marrow transplantation. As the D6S2878 STR is an old entity, it may also be used to study other populations of primate species.
The evolutionary stability of this microsatellite has been a matter of debate (Epplen et al. 1997. Hum Genet 99: 399-406; Riess et al. 1990. Immunogenetics 32: 110-6; Bergstrom et al. 1999. Am J Hum Genet 64: 1709-18; Trtkova et al. 1995. Mol Phylogenet Evol 4: 408-19; Maueler et al. 1999. Gene 226: 9-23). The HLA-DRB7 associated D6S2878 allele is especially remarkable, as this repeat has the shortest (GT)x as well as the (GA)y part and is highly stable in length, showing no polymerase slippage at all. This is in accordance with the fact that short and/or interrupted repeats are more stable than long, uninterrupted dinucleotides (Petes et al. 1997. Genetics 146: 491-8; Wierdl et al. 1997. Genetics 146: 769-79; Jin et al. 1996. Proc Natl Acad Sci USA 93: 15285-8). To what extent the repeat composition may have a direct or indirect influence on the low mutation rate of the adjacent exon 2 segment of the DRB7 pseudogene is not well understood at present. It has been proposed, for example, that microsatellites near genes may increase and probably also decrease local mutation rates (Vowles and Amos 2004. PLoS Biol 2: E199). Interestingly, exon 2 of the DRB7 pseudogene, present on the only shared DR region configuration of humans and chimpanzees, is highly conserved between both species. Moreover, the D6S2878 sequence is completely identical (FIG. 1). It has been suggested that the intron 2 segment containing the (GT)x(GA)y repeat may bind a zinc-dependent protein and forms non B-DNA structures; thus, functionality of these so-called ‘junk’ DNA sequences cannot be ruled out and should be subjected to further analysis (Maueler et al. 1999. Gene 226: 9-23).
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TABLE 1







Mamu-DRB haplotypes defined by exon 2 sequencing and D6S2878 genotyping




















1st DRB 
6th DRB 


2nd DRB 

3rd DRB 

4th DRB 

5th DRB 



hapl
locus
locus
STR
STR
locus
STR
locus
STR
locus
STR
locus
STR





 1a
DRB1*0406

205, 207, 211

DRB5*0301
169








 1b
DRB1*0411

223

DRB5*0304
153








 2
DRB3*0403

192

DRB*W305
227








 3a
DRB1*0412

199

DRB*W3701
235








 4a
DRB1*1010

205

DRB6*0121
216








 5a
DRB3*0408

203

DRB*W404
293








 6a
DRB3*0411

190

DRB*W314
219








 7b
DRB4*0102

291

DRB5*0306
173








 8 b
DRB1*0321

181

DRB1*0322/3
209








 9 b
DRB1*0321

181

DRB1*0322/3
209
DRB1*1003c
175?






10 b
DRB1*0309

229

DRB*W2507
181
DRB6*0109
?






11a
DRB1*0303

195

DRB1*1007
197
DRB6*0103
(202-206)






11b
DRB1*0312

195

DRB1*1007
201
DRB6*0103
?






11c
DRB1*0306

226

DRB1*1007
197
DRB6*0103
?






11d
DRB1*0306

226-230

DRB1*1003
199-
DRB6*0107
182












213








12
DRB1*0309

233-247

DRB6*0101
?
DRB*W201
263 (261, 267)






13
DRB6*0112

214

DRB*W2501
218?
DRB*W2002
218
DRB6*0106?
205?




14
DRB6*0114

218

DRB*W303
193
DRB*W401
187






15a
DRB1*0403

229

DRB6*0107
184
DRB*W501
?
DRB6*?
208




15b b
DRB1*0403

229

DRB6*0107
183
DRB*W502
?
DRB6*0120
208




16
DRB1*0404

225

DRB6*0102
164
DRB*W307
195
DRB*W702
188




17
DRB1*0701

197

DRB3*0405
237
DRB5*0303
169
DRB6*0123
184




18a a
DRB1*0326

214

DRB1*07032
211
DRB6*0124
170
DRB*W2603
177
DRB6*0120
?


18b b
DRB1*0326

214

DRB1*07033
207
DRB6*0124
168
DRB*W2604
177
DRB6*0120
208


19a
DRB1*0318

181

DRB6*0105
190, 
DRB*W604
201, 205
DRB*W603
275, 
DRB6*0104
222


(220)





192



277
(+ALU)



19b
DRB1*0318

181

DRB6*0105
194
DRB*W604
197
DRB*W611
275
DRB6*01?
222


20 a
DRB3*0403

182

DRB*W402
185
DRB*W2701
227
DRB6*0116
206
DRB6*0107
182


21a
DRB6*0111

178

DRB*W606
211, 
DRB*W2104
211, 213
DRB*W2603
177, 
DRB6*0122
236








213,



187










197 








21b b
DRB6*0108

176

DRB*W606
213
DRB*W2104
213
DRB*W2604
177
DRB6*0122
232


22
DRB1*0310

241

DRB6*0118
210
DRB*W101
219, 223
DRB*W602
279
DRB*W609
219



DRB6*0106

192






(285)

(223)





aAnimals with Chinese,


banimals with Burmese origin.


cDRB1*1003 belong to haplotype 8 or 9;


( ) STR length observed in some (1-3) animals;


? STR not or rarely detected but presence of gene ascertained by sequencing;


205?, 175? STR's detected but not confirmed by sequencing













TABLE 2







HLA-DRB haplotypes defined by sequencing and D6S2878 genotyping













Haplotype
DRB1 locus
STR
2nd DRB locus
STR
3rd DRB locus
STR

















DR8
DR8a
DRB1*0801(03)
170 (172)







DR8b
DRB1*080302
176






DR1
DR1a/DR103
DRB1*010101/0103
154 (156)
DRB6*010101
136





DR1b
DRB1*010201
160
DRB6*0101
136





DR10
DRB1*100101
161
DRB6*0101
136




DR52
DR17a
DRB1*030101
172
DRB3*010101(2)
180





DR17b
DRB1*0301
178-184
DRB3*0202(01
208









(206, 210)





DR11a
DRB1*110101
182-192
DRB3*020201
206-218





DR11b
DRB1*110201
186
DRB3*020201
214





DR11c
DRB1*110401
188
DRB3*020201
208 (210)





DR12a
DRB1*120101
200
DRB3*010102
180





DR12b
DRB1*1201
206 (208)
DRB3*02(0201)
210, 212





DR12c
DRB1*120201
216
DRB3*030101
186





DR13a
DRB1*1301(01)
194
DRB3*0101(02)
180





DR13b
DRB1*1301(01)
194, 196
DRB3*02(0201)
204-214





DR13c
DRB1*1302(01)
188, 206-218
DRB3*0301/0101
186





DR14a
DRB1*140101
192
DRB3*0211
212





DR14b
DRB1*140101
186, 188, 196
DRB3*020201
210





DR14c
DRB1*140101
198
DRB3*0201
214




DR53
DR4a
DRB1*0401(01)
180, 182, 184
DRB4*(010101)
178, 180, 
DRB7*010101
135







(176, 182/4)





DR4b
DRB1*0402
194
DRB4*010101
176
DRB7*010101
135



DR4c
DRB1*0404
192 (196)
DRB4*010101
178
DRB7*010101
135



DR4d
DRB1*040701
184
DRB4*010101
178
DRB7*010101
135



DR4e
DRB1*0408
180
DRB4*010101
178
DRB7*010101
135



DR7
DRB1*070101
149
DRB4*010101
170, 176, 180
DRB7*010101/2
135



DR9
DRB1*09(0102)
170 (196)
DRB4*010101
180
DRB7*010101
135


DR51
DR15a
DRB1*150101
186
DRB5*0101(01)
180
DRB6*(0201)
154



DR15b
DRB1*150101
186, 190 (184, 188)
DRB5*0101(01)
184 (190)
DRB6*(0201)
152



DR15c
DRB1*150201
202-208
DRB5*0102
220
DRB6*(0201)
174



DR16
DRB1*160101
178
DRB5*?
184
DRB6*0202
144





( ) indicates STR lengths detected only once or twice













TABLE 3







Gene frequencies and numbers (#) of Mamu- and HLA-DRB


haplotypes tested by D6S2878








Mamu











gfb
HLA












hapla
#
(2n = 240)
hapla
#
gfc

















 1a
43
(4)d
0.104
DR8a
5
(2)d
0.039


 1b
4

0.004
DR8b
2
(2)



 2
20
(2)
0.017
DR1a-DR103
42
(20)
0.094


 3
1

0.004
DR1b
8
(2)



 4
2

0.004
DR10
4

0.013


 5
2

0.004
DR52





 6
2

0.004
-DR17a
34
(16)
0.111


 7
1

0.004
-DR17b
12
(6)



 8
3

0.004
-DR11a
25
(2)
0.134


 9
1

0.004
-DR11b
2
(2)



10
4

0.008
-DR11c
6
(6)



11a
32

0.063
-DR12a
1

0.023


11b
7

0.017
-DR12b
5
(2)



11a or


0.083
-DR12c
1




be



-DR13a
13
(6)
0.102


11c
7
(4)
0.004
-DR13b
16
(6)



11d
21

0.096
-DR13c
12
(4)



12
58
(2)
0.163
-DR14a
1

0.032


13
10

0.008
-DR14b
4
(2)



14
7
(4)
0.021
-DR14c
2
(2)



15a
7
(2)
0.033
DR53





15b
2

0.008
-DR4a
23
(6)
0.128


16
3

0.029
-DR4b
2
(2)



17
7

0.025
-DR4c
11
(6)



18a
3

0.004
-DR4d
2




18b
2

0.004
-DR4e
1




19a
5

0.071
-DR7
28
(12)
0.132


19b
3

0.013
-DR9
10
(2)
0.014


20
1

0.008
DR51





21a
20
(2)
0.050
-DR15a1
5

0.107


21b
2

0.004
-DR15a2
39
(16)














22
14
(2)
0.042
-DR15b
4
(2)













other
n.t.
0.092
-DR16
2
(2)
0.036













total
294

1.000
others
n.t.
0.035












total
322

1.000





aMhc-DRB haplotypes correspond to those defined in Table 1 and 2, respectively.


bGene frequencies (gf) of Mamu-DRB haplotypes of the rhesus macaque breeding colony.


cGene frequencies (gf) of caucasoid HLA-DRB haplotypes according to Marsh and coworkers (39).


d( ) refer to # of homozygous typing cells included in the total #.


eHaplotypes 9a and b could only be descriminated by D6S2878 genotyping and sequencing;


n.t. not tested.













TABLE 4







Patr-DRB haplotypes defined by exon 2 sequencing and DRB-STR genotyping

















hapl*
1st DRB locus
STR
2nd DRB locus
STR
3rd DRBlocus
STR
4th DRB locus
STR
5th DRB locus
STR





 1
DRB1*0205
214
DRB3*0214L
182








 2
DRB4*0201
138
DRB*W902
192, 194








 3
DRB1*02new
228
DRB3*0102L
186
DRB5*0101L
170






 4a
DRB1*0302
190 (184)
DRB3*0102
186
DRB6*0305
155






 4b
DRB1*0308
178
DRB3*0102
186
DRB6*0305
155






 4c
DRB1*0309
176
DRB3*0102
186
DRB6*0305
155






 5a
DRB1*0701
157
DRB4*0104
186
DRB7*0101
135






 5b
DRB1*0702
153
DRB4*0104
186
DRB7*0101
135






 6
DRB1*1001
168
DRB5*0310
174
DRB6*0108
145






 7
DRB1*0309
176
DRB6*0305
155
DRB*W903
190






 8a
DRB1*0201
207, 211, 
DRB3*0201
186
DRB5*0301
174
DRB6*0108
144






217 (209)










 8b
DRB1*0201
209
DRB3*0208
220
DRB5*0306
174
DRB6*0109
145




 8c
DRB1*0201
209
DRB3*0208
204
DRB5*0304
174
DRB6*0108
145




 8d
DRB1*0202L
226
DRB3*0209L
206
DRB5*0306L
195
DRB6*0109
145




 8e
DRB1*02new
211
DRB3*0208
212
DRB5*0311
172
DRB6*0109
145




 8f
DRB1*0204
205
DRB3*0201
188
DRB5*0301
174
DRB6*0108
144




 8g
DRB1*0204
205
DRB3*0208
208
DRB5*0101L
170
DRB6*0109
145




 8h
DRB1*0204
205
DRB3*0208
208 (200)
DRB5*0102
170
DRB6*0109
145




 8i
DRB1*0204
205
DRB3*0208
210
DRB5*0102L
170
DRB6*0109
145




 8j
DRB1*0204
221, 225
DRB3*0208
212
DRB5*0101L
170
DRB6*0109
145




 9a
DRB1*0302
182
DRB3*0102
186
DRB5*0312
174
DRB6*0305
155




 9b
DRB1*0307
164, 174
DRB3*0208
186
DRB5*0301
174
DRB6*0305
155




 9c
DRB1*0307
166
DRB3*0102
186
DRB5*0306
174
DRB6*0305
155




 9d
DRB1*0307
172
DRB3*0102
186
DRB5*0102
170
DRB6*0305
155




10
DRB1*1001
170
DRB3*0208
208
DRB5*0310
174
DRB6*0108
155




11
DRB1*0311
184
DRB3*0208
202
DRB5*0307
174
DRB*W901
190




12
DRB1*0305
166, (168)
DRB3*0208
214, 218, 
DRB5*0310
174
DRB6*0108
145
DRB6*03?
155






222, (208)








13
DRB1*0201
205
DRB3*0208
202
DRB5*0307
174
DRB6*0108
145
DRB6*0305
155





*Thirteen region configurations (1-13) could be defined.


Letter surfixes (for example 7a-7k) indicate that a region configuration displays allelic polymorphism and can thus be split in different haplotypes.


Data in parentheses indicate STR lengths detected only once or twice













TABLE 5







Mafa-DRB haplotypes defined by exon 2 sequencing and DRB-STR genotyping















hapl
1st DRB locus 
STR
2nd DRB locus 
STR
3rd DRB locus 
STR
4th DRB locus
STR





 1
DRB1*0306
193
DRB5*0309
227
DRB*W6501
225
DRB6*0112
188


 2
DRB*W405
211
DRB*W2504
209
DRB6*0114
226




 3
DRB1*0309
209
DRB*W2001
283
DRB6*0107
?




 4
DRB*W6601
193
DRB*W2001
274
DRB6*0108
204




 5
DRB1*0411
229
DRB*W360202
229
DRB6*0115
214




 6
DRB1*0312
247 (239, 251)
DRB*W2502
185
DRB6*0107
208




 7
DRB1*0313
211
DRB*W3601
227
DRB6*0105
210
DRB*W102?
304


 8
DRB1*0314
181
DRB1*0315
211
DRB6*0112
176




 9
DRB1*0316
195
DRB*W4001
203
?
181?




10
DRB1*0317
193 (195)
DRB*W601
203
DRB*W2001
274
DRB*W6701
189 (195)


11
DRB1*0317
193
DRB*W601
203
DRB6*0106
?




12
DRB1*0401
189
DRB5*0303
169
DRB4*0102
231
DRB6*011302
182


13
DRB1*0401
189 (187)
DRB5*030101
169
DRB*W303
247




14
DRB1*0401
189
DRB5*030101
169
DRB4*0101?
255, 259




15
DRB1*0403
207
DRB*W3701
241
DRB6*011301
?




16
DRB1*0403
201
DRB*W3701
225
?





17
DRB1*0704
191
DRB*W605
281
DRB1*0308
234




18
DRB1*0704
?
?
172?
DRB*W5301
219




19
DRB1*1002
218, 208, (204)
DRB*W4901
259, 263, (265)
DRB6*0109
184




20
DRB3*0401
239
DRB5*0306
169
DRB6*0110
186




21
DRB*W6801
209
DRB6*0111
178






22
DRB*W2101
227 (229)
DRB6*0101
204 (206)
DRB*W501?
189(221)





Data in parentheses are STR length observed in some (one or two) animals.


Question marks indicate STRs not detected or rarely detected but presence of gene ascertained by sequencing.


172?, 181? Indicate STRs detected but not confirmed by sequencing.


DRB4*0101? And DRB*W501? detected on cDNA, but not on gDNA most likely due to primer inconsistency.













TABLE 6







Mafa-DRB haplotypes of a cynomolgus family (FIG. 10) defined by exon 2 sequencing


and DRB-STR typing













hapl
1st DRB locus
STR
2nd DRB locus
STR
3rd DRB locus
STR





a
DRB1*0403
201
DRB*W3701
225
?



b
DRB1*0704
191
DRB*W605
281
DRB1*0308
234


c
DRB1*0312
247 (239, 251)
DRB*W2502
185
DRB6*0107
208


d
DRB1*0401
189
DRB5*030101
169
DRB4*0101?
255, 259


(e*
DRB5*0305)







f
DRB1*0411
229
DRB*W360202
229
DRB6*0115
214


g
DRB*W2101
227 (229)
DRB6*0101
204 (206)
DRB*W501?
189 (221)


g*
DRB1*1002
218, 208, (204)
DRB*W4901
259, 263, (265)
DRB6*0109
184





Data in parentheses are STR length observed in some (one or two) animals.


Question marks indicate STRs not detected or rarely detected but presence of gene ascertained by sequencing.


172?, 181? Indicate STRs detected but not confirmed by sequencing.


DRB*W501? detected on cDNA, but not on gDNA most likely due to primer inconsistency.


*haplotype e only determined by cDNA typing






INCORPORATION OF SEQUENCE LISTING
Incorporated herein by reference in its entirety is the Sequence Listing for the application. The Sequence Listing is disclosed on a computer-readable ASCII text file entitled “Sequence_Listing_294-368_PCT_US_RCE_CON.txt,” created on Jul. 10, 2015. The sequence.txt file is 34 kilobytes in size.

## Claims

1. Method for detecting a DRB haplotype in a sample derived from a primate comprising nucleic acid from a primate cell, said method comprising:
amplifying at least an intron 2 microsatellite-containing part of at least three DRB-genes from said nucleic acid, wherein said amplification comprises providing said sample with a primer pair that spans the region containing said intron 2 microsatellite, wherein the primer pair comprises a first primer having a nucleotide sequence selected from a conserved region spanning the 3′ end of exon 2 and the 5′ start of intron 2, and a second primer the nucleotide sequence of which corresponds to a region comprising conserved sequences within the range of 5 to 60 nucleotides from the microsatellite;
detecting the type of intron 2 microsatellite present in each of said at least three DRB-genes, wherein said type of intron 2 microsatellite is determined based on the length of said microsatellite; and
determining the DRB haplotype from said type of intron 2 microsatellite present in said at least three DRB-genes, wherein the DRB haplotype is defined by the number of DRB genes and the specific DRB alleles of said DRB genes.

2. Method according to claim 1, wherein the DRB haplotype is determined by comparing the detected types of intron 2 microsatellite with a reference.

3. Method according to claim 2, wherein said reference comprises a database of DRB haplotypes correlated with intron 2 microsatellite types.

4. Method according to claim 1, wherein one or more of the primers used is labelled.

5. Method according to claim 1, wherein the nucleic acid from a primate cell comprises genomic DNA.

6. The method according to claim 1, wherein the microsatellite is the D6S2878 microsatellite.