Single Nucleotide Polymorphism (SNP)-Based Loss of Heterozygosity (LOH) Testing by Real Time PCR in Patients Suspect of Myeloproliferative Disease

During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n = 6 25–50% and n = 6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25–50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci

Cross table of <i>JAK2</i> region loss of heterozygosity results of the <i>JAK2V617F</i>-positive patient cohort, generated by the Short Tandem Repeat (STR) assay and the Single Nucleotide Polymorphism (SNP) based assay.

A<p> =  patient (pt) 5, 7, 9, 10, 11 and 12,</p>B<p> =  pt 3*, ^C =  pt 1, 2 and 6**.</p>*<p>LOH observed for a small region of the <i>JAK2</i> gene, in proximity of the <i>JAK2V617F</i> codon.</p>**<p>rs7869592 equivocal, other 4 SNPs indicative of ROH.</p><p><i>JAK2</i>LOH  =  loss of heterozygosity on the <i>JAK2</i> region, <i>JAK2</i>ROH  =  retention of heterozygosity on the <i>JAK2</i> region.</p

Cross table of <i>JAK2</i> region loss of heterozygosity results of the patients suspect of MPN cohort, generated by the Short Tandem Repeat (STR) assay and the Single Nucleotide Polymorphism (SNP) based assay.

D<p> =  patients 15, 39, 41, 44 and 61.</p><p><i>JAK2</i>LOH  =  loss of heterozygosity on the <i>JAK2</i> region, <i>JAK2</i>ROH  =  retention of heterozygosity on the <i>JAK2</i> region. MPN  =  myeloproliferative neoplasm.</p

Visualization of FAM/VIC ratios generated by representative SNP rs3780378

<p><b>A.</b>Scatterplot of SNP rs3780378 analyses of paired EDTA-blood and formalin-fixed paraffin-embedded (FFPE)-tissues of 93 patients (12 patients from <i>JAK2V617F</i>-positive cohort and 81 sample pairs from the MPN suspect patient cohort). X-axis: Delta Rn VIC (T  =  Thymine), Y-axis: Delta Rn FAM (C  =  Cytosine). White squares (bl-M1&2)  =  EDTA-blood samples amplified using ABI7500FAST machines 1 and 2, grey squares (bl-M3)  =  EDTA-blood samples amplified using ABI7500FAST machine 3, white triangles (par-M1&2)  =  FFPE-tissue samples amplified using ABI7500FAST machines 1 and 2 and grey triangles (par-M3)  =  FFPE-tissue samples amplified using ABI7500FAST machine 3. <b>B.</b> Representative box-and-whisker plot generated using the SNP rs3780378 VIC/FAM ratios of the EDTA-blood samples of all heterozygous patients from the patient cohort analyzed with ABI7500FAST machines 1 and 2. Stars represent extremes (>3× IQR  =  <i>JAK2</i>LOH).</p

Distribution of Short Tandem Repeat (STR) and Single Nucleotide Polymorphism (SNP) markers on the <i>JAK2</i> region, along part of chromosome 9p.

<p><b>A.</b>SNP (rs#) and STR (D9S#) marker loci within a 0.5 Mbp spanning genomic region harboring the <i>JAK2</i> gene (<i>JAK2</i> region). <i>RCL1</i> =  RNA terminal phosphate cyclase-like 1 gene, <i>JAK2</i> =  Janus Kinase 2 gene, <i>INSL4/6</i> =  insulin-like 4 and 6 genes and <i>RLN2</i> =  relaxin 2 gene. The arrow indicates the approximate position of <i>JAK2V617F. </i><b>B.</b> STR and SNP results of <i>JAK2</i> region of 16 patients (pt). PV =  polycythemia vera, NC  =  non-classified myeloproliferative neoplasm. The boxes represent all heterozygous markers. Black boxes  =  LOH observed for this marker, white boxes  =  retention of heterozygosity observed for this marker and striped boxes  =  equivocal for this marker, homozygous markers are not shown.</p

Cross table of equivocal and non-informative results, generated by the Short Tandem Repeat (STR) assay and the Single Nucleotide Polymorphism (SNP) based assay.

E<p> =  pt 32, 55 and 93,</p>F<p> =  pt 4 and 38,</p>G<p> =  pt 8 and 68,</p>H<p> =  pt 13, 20, 22, 24, 25, 59 and 69.</p><p><i>JAK2</i>LOH  =  loss of heterozygosity on <i>the JAK2</i> region, <i>JAK2</i>ROH  =  retention of heterozygosity on the <i>JAK2</i> region. Results were indicated as non-informative due to homozygous STR/SNP profiles in the formalin-fixed paraffin-embedded tissues. NA  =  not applicable.</p

Panel of selected Single Nucleotide Polymorphisms (SNPs).

<p>SNP-ID (hCV)  =  Celera SNP ID. SNP-ID (rs)  =  reference SNP ID number. Base (9p)  =  the nucleotide position on chromosome 9(p). The particular nucleotide variation is referenced in “SNP”-column. Minor allele frequencies are indicated for different populations: CEU, CEPH (Centre d’Etude du Polymorphisme Humain) from Utah; CHB, Chinese from Beijing; JPT, Japanse from Tokyo and YRI, Yoruba from Ibadan Nigeria.</p>*<p>SNP rs7862852 is excluded from the panel of recommended SNPs. NA  =  not applicable.</p