Pyrosequencing technology in Hematolgy

Pyrosequencing technology in Hematology – reliable sequence-based analysis for a demanding speciality

Pyrosequencing technology provides reliable genetic analysis in the study of a wide range of hematological conditions:

• Quality-controlled detection and quantification of mutations and SNPs
• Quantification of multiple, individual CpG methylation sites, with quality control of bisulfite treatment
• Rapid sequencing of informative regions
  

Filling the demand for flexible technology

The field of hematology is broad, including the study of blood, the blood-forming organs, and blood disorders. The genetic basis for blood disorders and their treatment includes:

• Mutations associated with errors in clotting, structural changes in hemoglobin etc.
• Mutations associated with neoplasms
• SNPs that affect drug response
• Changes in methylation of genetic control regions leading to neoplasms

The treatment of hematological neoplasms can also involve bone-marrow transplantation, with the need for sequence-based HLA-typing.

Such studies place great demands on methods that can detect polymorphisms and mutations, quantify levels of mutations in mixed populations in the study of minimal residual disease, quantify changes in methylation levels, and even sequence informative regions.

Pyrosequencing technology offers such flexibility and this is illustrated using the examples shown below. Pyrosequencing technology has also been used to sequence HLA loci.

Mutation detection and Allele Quantification: JAK2

The V617F mutation of the tyrosine kinase JAK2 gene is widespread in chronic myeloproliferative disorders, such that JAK2 mutation testing may rapidly become a frontline test. The sensitivity of the Pyrosequencing JAK2 assay enables accurate quantification of the mutation in minor subpopulations of blood or bone marrow myeloid cells. This assay is useful, for example, in laboratory studies and clinical trials testing new therapeutic agents for myeloproliferative diseases harboring the JAK2 V617F mutation. Further details of the detection and quantification of this mutation can be found in the literature listed in Table 1.

Figure 1. Pyrogram® traces of JAK2 analyses. The sequence read is (G/T)TCTGTGG. The shaded area indicates the mutation site. The G peak in the shaded box varies between 0 and 1 (normalized units), whereas the T peak varies between 1 and 2, since the subsequent nucleotide in the sequence is T. Top: healthy control; middle: Philadelphia chromosome negative CML, normal G is 40% and mutant allele is 60%; bottom: HEL cell line showing 100% mutant T allele. Source: Jelinek et al., Blood, 106 (10) 3370-3

PyroMark Research-Use-Only Tests 

QIAGEN offers a range of Research-Use-Only tests including several that are relevant to hematology:

Detection of mutations and SNPs

K-RAS - the analysis of mutations in codons 12, 13 and 61
MTHFR genotyping C677T variant
HFE - mutations H63D and S65C (exon 2) and C282Y (exon 4)

Quantification of CpG methylation levels

MLH1 (control region)
CDKN2A (p16; control region)
MGMT (control region)
LINE-1 (a region that can be used as a marker for global methylation)