Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-30
2002-05-07
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06383747
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention broadly relates to the field of molecular genetics. More specifically, the invention relates to methods for genetic analysis of DNA sequences corresponding to ancestral haplotypes of multigene complexes such as the Human Major Histocompatibility Complex and fragments thereof.
The Human Major Histocompatibility Complex (MHC) is a region of chromosomal DNA which plays a key role in the immune system and influences diverse functions and diseases. The MHC contains multiple polymorphic and duplicated genes (Zhang et al, 1990). At the centromeric end of the human chromosome on which the MHC is found there are genes such as HLA DR, DQ and DP which encode the Class II products involved in specific antigen presentation. At the telomeric end of the chromosome there are multiple genes.uch as HLA-A, B and C which encode the Class I products recognised by cytotoxic T cells. In the central region there are genes for amplifiers and mediators such as C4, C2, Bf and tumour necrosis factors (TNF-a, TNF-b), together with a heterogeneous collection of at least ten other genes (Sargent et al, 1989; Spies et al, 1989). Although only some of these genes have been sequenced it is already obvious that there is no single gene family, in that the products vary substantially in their structure. Genes such as heat shock protein 70 may possibly have a role in antigen processing prior to presentation. The products of some of the other genes may possibly be involved in intercellular adhesion, and perhaps some could influence immune responses as accessory molecules (Banerji et al, 1990). The B144 gene, together with the complement and TNF genes, could possibly play a role in the regulation of antibody production (French and Dawkins, 1990). Although more detailed information is required, it is possible that the MHC could be thought of as a chromosomal region containing heterogeneous genes which in various ways regulate and direct immunological responses, whether mediated by antibody, T cells or other effector mechanisms.
For many years it has been known that the MHC contains genes which influence the susceptibility to many diseases (Dawkins et al, 1983). Products of the human MHC are also intimately involved in tissue rejection, wherein transplanted tissues bearing MHC products which are non-identical to the transplant recipient are recognised as foreign by the immune system and rejected.
Because of the practical importance of transplantation, much effort has been spent in “tissue typing” within the MHC to ascertain whether candidate tissues for transplantation carry the same MHC ancestral haplotype as the intended recipient. Most of this typing has been by serological analysis focusing on the DR and DQ HLA class II gene products, the HLA B class I gene products, as well as other markers.
Current tissue typing techniques are subject to a degree of ambiguity which is inherently associated with serological determinations as well as time delays associated with such analysis. There is additionally a need for further “markers” in order to assess the compatibility or otherwise of tissue for transplantation.
“Tissue typing” is also important in assessing susceptibility and resistance to disease as mentioned above. Where a patient's susceptibility to autoimmune or other diseases can be detected at an early stage, appropriate therapeutic regimes, and counselling can be implemented before disease progression with attendant advantages.
Ancestral haplotypes are DNA sequences from multigene complexes such as MHC. The ancestral haplotypes of the MHC extend from HLA B to HLA DR and have been conserved en bloc. These ancestral haplotypes and recombinants between any two of them account for about 73% of ancestral haplotypes in our caucasian population. The existence of ancestral haplotypes implies conservation of large chromosomal segments. These ancestral haplotypes carry many MHC genes, other than the HLA, which may be relevant to antigen presentation, autoimmune responses and transplantation rejection. Tissue typing is an analysis of the combination of alleles encoded within the MHC. Many of these allelic combinations can be recognised as ancestral haplotypes. Other multigene complexes containing ancestral haplotypes include the lipoprotein gene complex and the RCA complex
This invention stems from characterisation of DNA corresponding to ancestral haplotypes. The inventors have surprisingly found that the DNA sequences corresponding to a particular ancestral haplotype are identical within that ancestral haplotype, whereas DNA sequences between ancestral haplotypes are very polymorphic. Each ancestral haplotype, therefore, possesses a unique nucleotide sequence which can be exploited in assigning ancestral haplotypes. Accordingly, genetic analysis and assignment of ancestral haplotype maybe readily conducted according to the methods of this invention. This is particularly important where identity at the genomic sequence level may be necessary for the most desirable outcome following grafts and transplants such as in bone marrow grafting (Christiansen et al, 1991). The genetic analysis of the present invention provides the ability to “match” ancestral haplotypes between individuals or to “type” ancestral haplotypes by a comparison to a reference panel of ancestral haplotypes.
DESCRIPTION OF THE INVENTION
In accordance with the first aspect of this invention, there is provided a method for genetic analysis comprising comparing a first DNA sequence corresponding to an ancestral haplotype or recombinant thereof of a multigene complex, such as the MHC, with one or more reference DNA sequences each corresponding to a separate ancestral haplotype so as to establish identity or non-identity there between and thus an assignment of ancestral haplotype of said first DNA sequence.
DNA sequences which may be compared in assigning ancestral haplotype may comprise the DNA sequence of any multigene complex such as but not limited to the lipoprotein gene complex, the RCA complex and the MHC complex. Preferably the multigene complex is the MHC such as HLA C between HLA B and TNF+B144, and/or a DNA sequence corresponding to one or more haplospecific geometric elements of the human Major Histocompatibility Complex as defined hereinafter.
The present invention is directed to genetic analysis of the genomes of higher organisms such as from mammals, plants and insects. Preferred mammals are humans, livestock animals, companion animals and wild animals. Most preferably, the mammal is a human.
DNA sequences corresponding to ancestral haplotypes or recombinants thereof may be readily analysed by a number of techniques such as DNA sequence analysis, restriction fragment length polymorphism (RFLP), reaction with haplospecific oligonucleotide probes, heteroduplex analysis, primer directed amplification and other methods as are well known in the art. The genome itself may be subject to the analysis or via cDNA or mRNA.
In a preferred aspect of the present invention, the comparison is by the use of oligonucleotide probes which may also be labelled with a reporter molecule or a primer to direct amplification.
According to this preferred aspect, there is provided a method for matching a particular ancestral haplotype in the genome of two or more individuals of a higher organism, said method comprising contacting a region of the genome or fragment or portion thereof from each individual with an oligonucleotide probe which hybridises to at least one complementary sequence within the ancestral haplotype and comparing the extent of hybridisation to match the non-identity or identity of the ancestral haplotype.
Another preferred aspect of the present invention provides a method for matching a particular ancestral haplotype in the genome of two or more individuals of a higher organism, said method comprising contacting a region of the genome or fragment or portion thereof from each individual with at least one oligonucleotide primer which hybridises to at least one complementary sequence within the ancestral
Abraham Lawrence Joseph
Dawkins Roger Letts
Arthur Lisa B.
Scully Scott Murphy & Presser
The Immunogenetics Research Foundation Incorporated
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