Customer Publications in November and December 2021

         December 30, 2021

The articles we saw this November and December cited a wide range of applications of our product suite. The following publications feature usage of our SNP & Variation Suite, VSClinical, and VarSeq products. We see them being utilized to identify loci associated with facial eczema in New Zealand sheep, somatic mutation response impact, and assisting in estimating breast cancer risk across population bases.

Genomic Tools for the Identification of Loci Associated with Facial Eczema in New Zealand Sheep

Facial eczema (FE) is a significant metabolic disease that affects New Zealand ruminants. Ingestion of the mycotoxin sporidesmin leads to liver and bile duct damage, which can result in photosensitisation, reduced productivity and death. Strategies used to manage the incidence and severity of the disease include breeding. In sheep, there is considerable genetic variation in the response to FE. A commercial testing program is available for ram breeders who aim to increase tolerance, determined by the concentration of the serum enzyme, gamma-glutamyltransferase 21 days after a measured sporidesmin challenge (GGT21). Genome-wide association studies were carried out to determine regions of the genome associated with GGT21. Two regions on chromosomes 15 and 24 are reported, which explain 5% and 1% of the phenotypic variance in the response to FE, respectively. The region on chromosome 15 contains the β-globin locus. Of the significant SNPs in the region, one is a missense variant within the haemoglobin subunit β (HBB) gene. Mass spectrometry of haemoglobin from animals with differing genotypes at this locus indicated that genotypes are associated with different forms of adult β-globin. Haemoglobin haplotypes have previously been associated with variation in several health-related traits in sheep and warrant further investigation regarding their role in tolerance to FE in sheep. We show a strategic approach to the identification of regions of importance for commercial breeding programs with a combination of discovery, statistical and biological validation. This study highlights the power of using increased density genotyping for the identification of influential genomic regions, combined with subsequent inclusion on lower density genotyping platforms.

McRae KM, Rowe SJ, Johnson PL, Baird HJ, Cullen NG, Bixley MJ, Plowman JE, Deb-Choudhury S, Brauning R, Amyes NC, Dodds KG, Newman S-AN, McEwan JC, Clarke SM. Genomic Tools for the Identification of Loci Associated with Facial Eczema in New Zealand Sheep. Genes. 2021; 12(10):1560.

The Impact of Somatic Mutations upon the Response to Combination Therapy with Ruxolitinib and Interferon in MPN Patients

The Philadelphia-negative myeloproliferative neoplasms (MPN) are associated with driver mutations in JAK2CALR, and MPL genes. Non-driver mutations affect disease progression and treatment response. Combination therapy with pegylated interferon-alpha2 (IFN) and ruxolitinib has recently been shown to induce hematologic and molecular responses in patients (pts) with MPN. We studied 25 pts by targeted next generation sequencing (NGS) of 42 genes and investigated the impact of somatic mutations (mut) on treatment response.

Twenty-five MPN pts with polycythemia vera (PV) (n=16, all JAK2V617F) and myelofibrosis (MF) (n=9, JAK2V617F (6), CALR (1), MPL (1), triple-negative (1)) participated in the study. NGS was performed on the Illumina platform on DNA from peripheral blood at baseline and after 24 months of combination therapy. Data were analyzed using Biomedical Genomics workbench and VarSeq. Variants with coverage <100x, variant allele frequency (VAF) <1%, introns and germline variants, and SNPs with minor allele frequency >1% were excluded. A mut with VAF <1% in either a pre- or post-treatment sample was retained if VAF was >1% in the paired sample. However, a mut with a number of mutated reads below 7 was considered absent. Only pathogenic mut were reported. Statistical analysis was done using either chi-square test or Wilcoxon signed rank/rank sum test in R 4.0.2. A p-value <0.05 was considered significant. The ELN and IWG-MRT response criteria were used.

At baseline and 24 months, PV-pts had a mean number of mut of 2.3 and 2.4, and MF-pts 2.2 and 3.0, respectively. In all pts, 0 and 1 (0/4%) patient had no mut, 10 and 7 (40/28%) had 1 mut, 10 and 9 (40/36%) had 2 mut, and 5 and 8 (20/32%) had ≥ 3 mut at baseline and 24 months, respectively.

In PV, 1 (6%) achieved CR and 3 (19%) achieved PR and 12 (75%) achieved NR. In MF, 4 (44%) achieved CR and 1 (11%) achieved PR and 4 either NR, PD, SD or CI. There was no association between pts achieving CR/PR or NR and median number of mut at baseline (CR/PR: 1, range 1-3) or (NR: 2, range 1-8), (p=0.28). Excluding driver mut, there was still no significant association.

In all JAK2V617F positive pts, the median JAK2V617F allele burden (%JAK2V617F) decreased from 37% (range: 1.9-95) at baseline to 22.5% (range: 0-85) at 24 months. In pts achieving CR or PR (n=7), the reduction in %JAK2V617F was greater (median: 42% to 18%, p<0.05) compared with pts achieving NR (n=12) (median: 30% to 22%, p<0.005).

Stratified according to molecular response (MR) (n=4) or non-MR (n=13), median % JAK2V617F at baseline was 40 (21-95) and 43 (42-70) in non-MR and MR, respectively, and 31 (11-85) and 13 (1.8-25) in non-MR and MR, respectively during treatment. At baseline, pts achieving MR had no non-driver mut and pts achieving non-MR had a median number of non-driver mut of 1 (range: 0-7) (p<0.02). Eight pts were not evaluable because they were either CALR or MPL positive, triple-negative, or had a baseline JAK2V617F ≤ 20%.

In all pts, there were 30 non-driver mut in 15 genes at baseline and 40 non-driver mut in 19 genes at 24 months of therapy. At baseline and at 24 months, TP53 occurred in 6 and 6 (24%), TET2ASXL1RUNX1, or SF3B1 in 2 and 3 (8/12%), and CBLDNMT3A, or SRSF2 in 2 and 2 (8%) pts, respectively. Eleven new non-driver post-treatment mut occurred in 9 pts and were more prevalent in pts with MF (6/9, 66%) compared to pts with PV (3/16, 19%), p<0.02. However, the VAF was low (median 1.2%, range 1.02% – 3.6%) and none of the new 11 post-treatment mut appeared in the same gene.

Earlier studies have shown an association between poor response to IFN and number of mut or high molecular risk mut. In this study, the presence of non-driver mut was associated with a significantly poorer molecular response. However, there was no association between the presence or type of non-driver mut and clinico-hematologic response. At 24 months, there was an evolution of subclones with a significantly higher number of new subclones appearing in pts with MF compared to pts with PV. However, the VAF was low and none of the acquired mut appeared in the same gene contradictory to another study showing an association between treatment-emergent mut in DNMT3A and treatment with IFN. In conclusion, these data show that a clinico-hematologic response is achievable during combination therapy despite the presence of non-driver mutations at baseline.

Vibe Skov, Anders Lindholm Sørensen, Trine Alma Knudsen, Mads Emil Bjørn, Christina Ellervik, Morten Kranker Larsen, Christina Schjellerup Schjellerup Eickhardt-Dalbøge, Sarah Friis Christensen, Mads Thomassen, Torben A Kruse, Hans C. Hasselbalch; The Impact of Somatic Mutations upon the Response to Combination Therapy with Ruxolitinib and Interferon in MPN Patients. Blood 2021; 138 (Supplement 1): 3589. doi:

Population-based estimates of breast cancer risk for carriers of pathogenic variants identified by gene-panel testing

Population-based estimates of breast cancer risk for carriers of pathogenic variants identified by gene-panel testing are urgently required. Most prior research has been based on women selected for high-risk features and more data is needed to make inference about breast cancer risk for women unselected for family history, an important consideration of population screening. We tested 1464 women diagnosed with breast cancer and 862 age-matched controls participating in the Australian Breast Cancer Family Study (ABCFS), and 6549 healthy, older Australian women enroled in the ASPirin in Reducing Events in the Elderly (ASPREE) study for rare germline variants using a 24-gene-panel. Odds ratios (ORs) were estimated using unconditional logistic regression adjusted for age and other potential confounders. We identified pathogenic variants in 11.1% of the ABCFS cases, 3.7% of the ABCFS controls and 2.2% of the ASPREE (control) participants. The estimated breast cancer OR [95% confidence interval] was 5.3 [2.1–16.2] for BRCA1, 4.0 [1.9–9.1] for BRCA2, 3.4 [1.4–8.4] for ATM and 4.3 [1.0–17.0] for PALB2. Our findings provide a population-based perspective to gene-panel testing for breast cancer predisposition and opportunities to improve predictors for identifying women who carry pathogenic variants in breast cancer predisposition genes.

Southey, M.C., Dowty, J.G., Riaz, M. et al. Population-based estimates of breast cancer risk for carriers of pathogenic variants identified by gene-panel testing. npj Breast Cancer 7, 153 (2021).

We at Golden Helix enjoy seeing all the ways our software suite is utilized throughout the world. With that, we encourage all of our users to submit their research, whatever application, to our Innovation Awards. Show us how you use our products and you might receive the opportunity to highlight your research via a webcast and blog post.

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