Clinical Applications of Genetic Phasing (haplotyping)
1. Bone marrow transplantation between genetically unrelated individuals
Both the Perth group (Dawkins, Christiansen, Witt) and the Seattle group led by E.Petersdorf have shown repeatedly over the years that HLA haplotype matched unrelated donor/recipient pairs have less severe graft versus host disease than pairs that are allele matched across the HLA loci.
Given there are now in excess of 42 million donors worldwide, most of whom are not haplotyped there is a need to provide such information to obtain the best outcomes for patients. No such reproducible technology exists which can provide genetic phase across 3mb of DNA.
2. Drug metabolism
There is a family of polymorphic genes called CYP (cytochrome P) which are involved in the metabolism of many common pharmaceuticals. variation in these genes consists of single mutations, insertions and deletions, or a combination of these. Until definitive phasing is determined it is very difficult to determine what is occurring at the molecular level.
3. Cancer Genetics and Checkpoint Inhibitors.
Many genes which are involved in cancer development are known to be polymorphic. Again, to fully appreciate the clinical significance of these polymorphism accurate phasing is essential.
In addition, there is currently great interest in the role of checkpoint inhibitors to treat various types of human cancer. One such inhibitor is a monoclonal antibody to the CTLA-4 receptor (Ipilimumab) which has had good success in melanoma but not for all patients. Elucidation of phase in CTLA-4 in addition to biding studies with the CTLA-4 antibody will be informative. There will be many other monoclonal antibodies developed for various cancers in future.
4. Complex Multigenic Diseases including Autoimmune Diseases
Many multigenic disease patients particularly those with auto immune diseases will benefit greatly from genetic phasing. An example is type I diabetes where up to 50 polymorphic genes may be involved in the aetiology. The HLA system accounts for the majority of the genetic risk, and the importance of haplotypes has been demonstrated. However, haplotype information on the other polymorphic genes spread around the genome is scarce. Until this information becomes available no systems model is possible to explain the molecular events involved in disease pre-disposition.
5. Animal Genetics
Our technology is also applicable to some animal models.
For example, marbling in Wagyu beef has been demonstrated to be inherited as haplotypes. Our technology will provide this information in the absence of family studies.
Athletic sprinting or staying potential in thoroughbred horses has been demonstrated recently to be inherited as alleles in a series of polymorphic genes. HT technology will permit this information to be assembled in the absence of reference to the sire or dam.
Brian Tait has recently published a paper in the International Journal of Immunogenetics which deals more fully with his topic. This paper an be accessed through the following link:
https://www.researchgate.net/publication/357362445_The_importance_of_establishing_genetic_phase_in_clinical_medicine
Historical and Recent Comments on Haplotyping
The famous geneticist Craig Venter who along with Francis Collins published the first human genome sequence in 2001 said at the time
“The genome revolution is just beginning. Improving data quality is crucial because if a human genome cannot be independently assembled then the sequence data cannot be sorted into the two sets of parental chromosomes or haplotypes. This process haplo- phasing will become one of the most useful tools in genomic medicine. Establishing the complete set of genetic information that we received from each parent is crucial to understanding the heritability, gene function regulatory sequences and our predisposition to disease”
These comments now have to be viewed in the light of next generation and long- range sequencing. It is possible now to provide, reproducibly, haplotypes of some 10-20 kb in length. However, this is well short of the 3-4 mb of the HLA system. The other point to stress is the role of non-coding RNA molecules in controlling gene expression. These long and short RNA molecules, which have been shown to be polymorphic, are in many cases megabases away from their target gene. To fully appreciate the role and interaction of these RNA molecules with their targets will require phasing across megabases. This may become possible with long range sequencing in the future, but not now. By contrast, HT technology has the potential to deliver this now.
Following are some recently published comments on the importance of genetic phasing in some of the key clinical areas:
Bone marrow transplantation
“The future of histocompatibility testing in support of allogeneic transplantation will incorporate consideration not only of the classical HLA genes, but also of functional variation that is linked to HLA loci on haplotypes, and KIR genes. Ultimately, the key to understanding the histocompatibility barrier in transplantation will require information on the content of the MHC region and the organization of that genetic variation on haplotypes”
Dr E Petersdorf 2016
In Chapter 10 Histocompatibility
Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation
Stephen J. Forman, Robert S. Negrin, Joseph H. Antin, and Frederick R. Appelbaum pub Wiley.
Second, there are also other circumstances where genetic phasing plays a role. For example, hematopoietic stem cell transplants using haplotype matched recipients perform better clinically than those using allele matched but haplotype mismatched patients [36]. To mention another example that applies methods akin to our own work, an interesting new proposal appeared recently in the field of preimplantation genetic diagnosis (PGD) for molecular disorders. ...
Cross-ethnic analysis of common gene variants in hemostasis show lopsided representation of global populations in genetic databases
Abdimajid Osman1,2* and Jon Jonasson2,
n BMC Medical Genomics 2022. 15:69
Pharmacogenetics
There is evidence that patients carrying specific HLA haplotypes are more vulnerable to form anti-drug antibodies and this may be useful to personalise treatment
Immunogenicity of Monoclonal Antibodies and the Potential Use of HLA Haplotypes to Predict Vulnerable Patients
Frontiers in Immunology 2022
Romy Mosch and Henk-Jan Guchelaar
Complex multigenic diseases
“Present findings address that these haplotype combinations 111/112, 111/121 and 122/122 of CAPN-10 SNP-43, -19 and -63 constitute unique DNA biomarker fingerprints toward susceptibility and risk for T2DM and MS among Egyptians when compared to other haplotype combinations reported in other populations of different ethnicity. To enhance the power of human evolution control nowadays, mutations and polymorphisms in target genes associated with human diseases should be well understood”
Association of CAPN10 haplotype combinations with type 2 diabetes mellitus and metabolic syndrome among Egyptians: pilot study—genotyping of three CAPN10variants
Shaymaa W. El Far, Heba Sh. Kassem, Amira M. Embaby, Abir A. Saad, Nader Mowafy and Medhat Haroun 2022
Egyptian Journal of Medical Human Genetics 23:26
Our data suggest that genotypes comprising specific pairs of RET haplotypes are associated with predisposition to HSCR either in a simple autosomal recessive manner or in an additive, dose dependent fashion RET genotypes comprising specific haplotypes of polymorphic variants predispose to isolated Hirschsprung disease.
RET genotypes comprising specific haplotypes of polymorphic variants predispose to isolated Hirschsprung disease
Salud Borregoa, et al, 2000
Journal of Medical Genetics volume 37 no.8
Animal Genetics
“Traditional analyses of a QTL on Bota 19 implicate a surfeit of candidates, but each is of marginal significance in explaining the deposition of healthy, low melting temperature fat within marbled muscle of Wagyu cattle. As an alternative approach, we have used genomic, multigenerational segregation to identify 14 conserved, ancestral 20 Mb haplotypes. These determine the degree and rate of marbling in Wagyu and other breeds of cattle.”
2017 Haplotypes for Type, Degree, and Rate of Marbling in Cattle Are Syntenic with Human Muscular Dystrophy Sally S. Lloyd, Edward J. Steele,1 Jose L. Valenzuela, and Roger L. Dawkins. International Journal of Genomics Volume 2017, Article ID 6532837
“Mitochondrial DNA (mtDNA) encodes the genes for respiratory chain sub-units that determine the efficiency of oxidative phosphorylation in mitochondria. The aim of this study was to determine if there were any haplogroups and variants in mtDNA that could be associated with athletic performance of Thoroughbred horses. The whole mitochondrial genomes of 53 maternally unrelated Australian Thoroughbred horses were sequenced and an association study was performed with the competition histories of 1123 horses within their maternal lineages. A horse mtDNA phylogenetic tree was constructed based on a total of 195 sequences (including 142 from previous reports). The association analysis showed that the sample groups with poor racing performance history were enriched in haplogroup L3b (p = .0003) and its sub-haplogroup L3b1a (p = .0007), while those that had elite performance appeared to be not significantly associated with haplogroups G2 and L3a1a1a (p > .05). Haplogroup L3b and L3b1a bear two and five specific variants of which variant T1458C (site 345 in 16s rRNA) is the only potential functional variant. Furthermore, secondary reconstruction of 16s RNA showed considerable differences between two types of 16s RNA molecules (with and without T1458C), indicating a potential functional effect. The results suggested that haplogroup L3b, could have a negative association with elite performance. The T1458C mutation harboured in haplogroup L3b could have a functional effect that is related to poor athletic performance”.
Association of low race performance with mtDNA haplogroup L3b of Australian thoroughbred horses
Lin, X et al 2017
Mitochondrial DNA part A
There are many other applications of gene phasing not mentioned here. Proteins are coded by bases in cis phase. It is therefore critical to establish phase in order to appreciate the effect of multiple mutations in a gene sequence