Single Chromosome Sequencing: An overview

In this exclusive eBook, Haplomic Technologies (HT) explores the questions: What is single chromosome sequencing, and why is it important?

Single chromosome sequencing (SCS) is the sequencing of chromosomes separated from a single cell using a microfluidic device.

To date, Haplomic Technologies (HT) scientists are the only ones to have achieved this. Essentially, the technique involves the lysis of metaphase cells and the collection of single chromosomes, and subsequent sequencing.

The technique involves using microfluidic channels. Although we have not routinely recovered all 46 chromosomes from a single cell, we have succeeded in sequencing to identify each of the 23 human chromosomes. This is important in the field of personalised medicine. It matters because alleles need to be identified, and gene interactions understood. Without allele information, it is impossible to do this; SCS plays a crucial role in assigning both alleles of a gene in an individual, without relying on linkage disequilibrium or family studies.

Recently, alleles have been described by linkage disequilibrium, and haplotypes refer to genetic changes within an allele

The concept of linkage disequilibrium and haplotype has changed since their original description some 100 years ago in the case of LD (1), and over 60 years in the case of haplotypes ⁽²⁾.

The two scientists involved, Rainard Robbins and Ruggero Ceppellini, stated their theories and presented them at meetings. In the first case, which involved the LD between genes, and in the case of haplotype Ceppellini invoked the use of the word haplotype to explain the observed LD between the alleles present in the data of the 3rd Histocompatibility Workshop. These alleles are inherited together from one parent, as a haplotype.

The meaning of LD and the word haplotype changed with the introduction of sequencing and the difficulty in identifying alleles at many important genes studied. LD now also refers to including single nucleotide polymorphisms shown to be in LD, which in turn define alleles. This is a problematic concept, since LD varies between ethnic groups. This has been addressed to some extent with next generation sequencing (NGS), which allows sequencing of larger DNA segments than was possible with conventional Sanger sequencing. However, many researchers examining long genes often focus only on sequencing the exons, assuming that exon changes can alter the function of the resulting molecule. This approach does not consider intron polymorphisms, which also contribute to the definition of alleles. This issue can be addressed with SCS, using carefully selected primers in a PCR-based assay.

The level of expression is important in determining the impact of mutations on gene function

Also, pertinent is the role of micro-RNA (miRNA) molecules and long noncoding RNA molecules (lncRNA) play a role in the level of expression of many genes. There is evidence that they are polymorphic, and until alleles are defined, it will be extremely difficult to have a full description of a patient’s risk of disease.

The role of haplotyping in personalised medicine

Personalised medicine is best described as the tailoring of treatment according to the available data concerning a patient’s genetic profile. This can be an individual’s response to a drug, the best-known example being B*5701 and adverse effects to the drug Abacavir ⁽³⁾. It also applies to an individual’s response to a monoclonal reagent used in transplantation or human cancers.

SCS ‘s role in personalised medicine

As described in an earlier publication on the Open Access Government website ⁽⁴⁾, the CYP genes are a family of genes which control the metabolism of many drugs. Although mutations have been associated with the deficient metabolism of several drugs there has been no serious effort made to define alleles. SCS has a definite role in assisting in defining alleles. The level of expression of the CYP genes is also important in defining the impact of mutations on function.

The miRNA and lncRNA need to be identified and their polymorphism defined. SCS has a role in this aspect as well.

The first iteration of the instrument will be aimed at the bone marrow (hemopoietic stem cell) transplantation field and haplotype matching for the HLA complex. At present, a patient’s HLA haplotypes are determined by family studies looking for a two-HLA-haplotype-matched bone marrow donor. In approximately 30% of cases this results in a positive outcome, the remaining 70% either being transplanted by a one haplotype family donor or seeking a perfect match amongst the bone marrow registries which comprise in excess of 40 million donors ₍₅₎ ( see world-wide marrow donor association), most of whom have some level of HLA typing but very few have been HLA haplotyped.

So, we have a situation where an HLA haplotyped patient is searching for the registries full of individuals who are not HLA haplotyped, despite evidence that HLA haplotyping is associated with improved outcomes compared with allele-matched transplants ⁽⁶⁾. We are now in a position to match unrelated transplant pairs for HLA haplotypes using SCS. Additionally, when appropriate family members are unavailable to assign HLA haplotypes, they can be assigned to a patient through SCS.

References

1. Robbins RB ,1918. Some applications of mathematics to breeding problems. III. Genetics 3, pp375-489.
2. Ceppellini (ed) 1968. Histocompatibility Testing 1968. Pub Munksgaard Copenhagen.
3. Mallal S et al 2008 New England Journal of Medicine] 358(6), pp.568–579.
4. Open Access Government | Government | Health & Social Care | Research
5. About WMDA | WMDA
6. Kitcharoen K et al, 2006. Human Immunology 67, 238–246.

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