Definitive Diagnostics
HAPLOTYPING
Reduction to Practice
New generation sequencing (NGS) technology has addressed many of the early problems of read length and accuracy, sample throughput and cost of sequencing. Recent developments in Nanopore technology promises to improve NGS even further. However NGS is almost exclusively performed on diploid DNA sample material which can only determine inferred haplotypes. Inferred haplotypes do not meet the basic requirements for the practice of personalized medicine, transplantation, disease risk analysis, pharmacogenomics etc.
Haplomic Technologies’ IP embodies the technical capability to separate chromosome pairs for the sequencing of each haplotype. These two sets of sequences reveal haplotypic phase (cis or trans configuration) and provide for the determination of definitive haplotypes.
The practice of definitive haplotyping is very much dependent on the development of high throughput - low cost enabling technology platforms for capturing single chromosome sourced material for sequencing. Currently there are no such technology platforms commercially available. However, increased research on the genomics and sequencing of single cells and technologies such as microfluidics hold great prospect for the future development of such enabling technology platforms. The exciting work done by Stephen Quake and his Stanford University team supports this view. (http://www.innovationnewsdaily.com/mit-transform-world-tech-1920/2)
In the interim, other techniques such as laser capture microscopy, flow cytometry and manual manipulation from metaphase cell preparations, while capital and/or labour intensive, are economically viable techniques for some applications where there are overwhelming clinical advantages for the determination of definitive haplotypes.
APPLICATIONS
Pharmacogenomics
Pharmacogenomics is a branch of personalized medicine that examines the inherited variations in genes that dictate drug response and explore the ways these variations can be used to predict whether a patient will have the desired clinical response to a drug, an adverse response, or no response at all.
Up until recently, there has been no simple and accurate method for determining how an individual patient or a population group will respond to a drug. Pharmaceutical companies have been essentially limited to developing drugs that suit the "average" patient.
Now Pharmaceutical companies are required to know what genes etc influence the pharmacodynamics of their drug to ensure that only those patients who derive clinical benefit will be prescribed the drug. Specifically the clinical trial phase of drug development programs will need to determine which haplotypes deliver the desired clinical response. The Physician may then determine to which haplotype group a patient belongs, and then prescribe a course of treatment based on which medications and dosages are most successful with those haplotypes.
Haplotyping is the only analysis that has the disciminating power to determine the unequivocal relationship between genetics and drug response.
Bone Marrow Transplantation
Haplotyping in general but in particular regarding genes of the Major Histocompatibility Complex (MHC) or HLA complex allows alleles at individual loci to be assigned as being either in cis or trans. This is important in transplantation particularly stem cell or bone marrow transplantation because it has been previously shown that donors and recipients matched for HLA haplotypes have a superior clinical outcome compared with those that are matched for alleles at individual HLA loci but not haplotype matched. This probably indicates that there are intervening genes that play a role in determining clinical outcome.
The majority of bone marrow or haematopoietic stem cell transplants are performed for leukaemia and other blood related diseases and are performed between unrelated individuals. The ideal donor is an HLA haplotype identical sibling and these provide the best clinical outcome. Haplotype identity is established by typing all family members. However only a minority of patients have such an HLA matched donor and are required to search worldwide the unrelated bone marrow registries. There are currently 16 million HLA typed donors but haplotypes have not been defined in these donors as they are recruited as individuals and hence no family studies are performed. Establishment of HLA haplotypes in unrelated donors would require access to family members and would increase the cost of recruitment to the registries by at least 400%.
As a result “HLA matched” unrelated donors are defined as being allele identical at several individual HLA loci without any understanding as to which alleles occur on which chromosome. The ability to haplotype and HLA match unrelated donors without access to family studies would represent a quantum breakthrough in the way donors and recipients are selected for unrelated bone marrow transplantation.
Disease Risk Analysis
Currently disease gene localization and polymorphism studies are performed using DNA from unrelated individuals which consists by definition of two inherited chromosomes representing the gene region under study. When disease related mutations or single nucleotide polymorphisms (SNPs) are described it is impossible to determine whether they are in cis or trans configuration. The ability to determine configuration is critical in this instance since without this information the sequence of the disease gene cannot be ascertained.
For example:
(a) Type 1 Diabetes
Recently the Type 1 Diabetes Consortium collected DNA worldwide from several thousand type 1 diabetic individuals and family members to establish the importance of HLA haplotypes in conferring disease. Alleles were established by conventional typing methods and then haplotypes constructed from the data. The combination of alleles which comprise these haplotypes are known to be important determinants of disease susceptibility.
The ability to assign haplotypes in the absence of family studies would reduce the cost of such studies by at least 80%. This concept of course applies to other gene systems other than HLA and has potential widespread.
(b) Nasopharyngeal Carcinoma (NPC)
Professor Malcolm Simons’ recent editorial “Nasopharyngeal Carcinoma [NPC] as a Paradigm of Cancer Genetics", describes the necessity of resolving diploid genomic DNA into the component two parental haploid sequences in order to definitively assign the haplotypes conferring major histocompatibility complex (MHC) risk for NPC. The strategy is applicable not only to the 100+ other diseases that have MHC-HLA genetic associations, but also to genome-wide search for definitive genetic risk in any disease. (Chin J Cancer; 2011; Vol. 30 Issue 2; www.cjcsysu.com)