TP53 Allelic Status in Myeloid Malignancies

Introduction TP53 is a tumor suppressor gene involved in regulating cell division and apoptosis in response to DNA damage. In hematologic malignancies, TP53 alterations are present in both myeloid and lymphoid malignancies. TP53 alterations including both sequence level mutations and deletions occur in 8-10% of de novo acute myeloid leukemia (AML), and are significantly enriched in patients with therapy-related myeloid neoplasms with a frequency of 25-40%. TP53 alterations are associated with complex karyotype, resistance to traditional cytotoxic chemotherapy and dismal outcome, and are well established poor prognostic markers for both AML and myelodysplastic syndrome (MDS). While both biallelic and monoallelic TP53 alterations are seen, biallelic TP53 alteration is more frequent in both AML and MDS. In the instances of monoallelic TP53 alterations, the remaining wild-type allele can be inhibited by the dominant negative effect of the mutant p53. The clinical implication of TP53 allelic status remains controversial. Biallelic TP53 alterations and/or high TP53 mutation variant allele frequency (VAF) are associated with older patients, complex karyotype, few co-occurring mutations and poor outcome in both AML and MDS. Other studies demonstrate that biallelic vs. monoallelic TP53 alterations or high vs. low TP53 VAF have similar prognosis in myeloid malignancies. Current European LeukemiaNet (ELN) recommends testing TP53 deletion using karyotype analysis and TP53 mutations by molecular testing. Methods: We performed a retrospective review of patients with myeloid malignancies, myeloid next generation sequencing (NGS) panel and cytogenetic tests performed at ARUP Laboratories. We identified 18 patients with myeloid malignancies and TP53 mutations. We used a combination of karyotype analysis, FISH, chromosomal microarray (CMA) and NGS to determine the TP53 allelic status. Results: 18 patients diagnosed with myeloid malignancies and TP53 mutations were identified. Among them, 6 were diagnosed with AML, 10 had MDS, one with CMML and one had post essential thrombocythemia myelofibrosis. Age range is from 31 to 78 with a median age 65.5 years. 23 TP53 mutations were identified among 18 patients. The majority (78%) of TP53 mutations are located in the DNA binding domain. Co-occurring mutations are uncommon in patients with TP53 mutations. 11 out of 18 patients did not have co-occurring mutations in other myeloid malignancy related genes. Three cases had 1, three cases had 2, and one case had 3 co-occurring mutations at the time of diagnosis. Karyotype analysis and FISH were performed on all 18 patients. CMA was performed on 9 patients. 17p abnormalities, defined by the deletion or copy-neutral loss of heterozygosity (CN-LOH) of 17p, were seen in 9 out of 18 cases. The 17p abnormalities in 8 out of these 9 cases were visible by karyotype. Case 11 had a CN-LOH identified by CMA which was cryptic by karyotype. Typical complex karyotype was seen in 16 out of 18 cases. Biallelic TP53 alterations are defined by either the presence of a TP53 mutation with a 17p abnormality (deletion or CN-LOH), or two TP53 mutations with similar VAF, or one TP53 mutation with VAF >50%. Biallelic TP53 alterations were seen more frequently compared with monoallelic TP53 alterations (14 vs. 4 patients) and enriched in older patients (Figure 1). All 6 AML patients had biallelic TP53 alterations. Outcome data is available in 10 patients including 8 patients with biallelic and 2 patients with monoallelic TP53 alterations. Patients with biallelic TP53 alterations have dismal outcome (Figure 2). Patient 7 represents an atypical patient with TP53 alteration in our study cohort. She was diagnosed with MDS at the age of 31. She had monoallelic TP53 mutation with a relatively low VAF and normal karyotype through her disease course. Her MDS never progressed and she remained in remission after transplant for more than four years and still doing well. Conclusion: In this study, we evaluated the TP53 allelic status in 18 patients with myeloid malignancies. Biallelic TP53 alterations are more frequent than monoallelic TP53 alterations, and are associated with older patients, and fewer co-occurring mutations. Biallelic TP53 alterations are associated with typical complex karyotype and dismal outcome. Our study supports the importance to differentiate between biallelic and monoallelic TP53 alterations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

SNP Alleles Associated With Low Bolting Tendency in Sugar Beet

The identification of efficient molecular markers related to low bolting tendency is a priority in sugar beet (Beta vulgaris L.) breeding. This study aimed to identify SNP markers associated with low bolting tendency by establishing a genome-wide association study. An elaborate 3-year field trial comprising 13 sugar beet lines identified L14 as the one exhibiting the lowest bolting tendency along with an increased survival rate after autumnal sowing. For SNP discovery following phenotyping, contrasting phenotypes of 24 non-bolting and 15 bolting plants of the L14 line were sequenced by restriction site-associated DNA sequencing (RAD-seq). An association model was established with a set of 10,924 RAD-based single nucleotide polymorphism (SNP) markers. The allelic status of the most significantly associated SNPs ranked based on their differential allelic status between contrasting phenotypes (p < 0.01) was confirmed on three different validation datasets comprising diverse sugar beet lines and varieties adopting a range of SNP detection technologies. This study has led to the identification of SNP_36780842 and SNP_48607347 linked to low bolting tendency and can be used for marker-assisted breeding and selection in sugar beet.

Allele frequencies of Ppd-D1a, Ppd-B1a, and Ppd-B1c of photoperiodic sensitivity genes in spring bread wheat varieties (Triticum aestivum L.) of various origin

Aim. To identify and evaluate allele frequencies of Ppd-D1a, Ppd-B1a, Ppd-B1c and Ppd-1 of the genotypes of spring bread wheat varieties from various climatic zones. Methods. DNA isolation, allele-specific PCR, electrophoresis in agarose and polyacrylamide gel, statistical analysis. Results. 137 varieties of spring bread wheat of various origin were detected to identify Ppd-1 genotypes of Ppd-D1a, Ppd-B1a and Ppd-B1c allele carriers. The results for the total sampling of the varieties under investigation and the sampling of Asian varieties yielded six different Ppd-1 genotypes in each. As for samplings of other regions, there were from two (Mexico) to four (Europe, the USA, Canada, Ukraine) Ppd-1 genotypes. In the total sampling of varieties, there was a high incidence (20.5 %) of genotypes, dominant only in allele Ppd-D1a, varying from 0 (Russia) to 85.0 % (Mexico). The incidence of the genotypes with monogenically dominant Ppd-B1a (7.3 %) or Ppd-B1c (5.1 %) in the total sampling, was considerably lower. These genotypes were most common for the sampling of the varieties from the USA and Canada (25.0 and 16.7 % respectively). Digenically dominant Ppd-D1a Ppd-B1a genotypes were found in the total sampling with relatively low incidence (7.3 %), and were notable for the varieties from Asia (33.4 %), Mexico (15.0 %), Ukraine (13.1 %), and Europe (3.1 %). The di genically dominant genotype Ppd-D1a Ppd-B1с was found only in the Japanese variety Konosu-25. Gene Ppd-A1 was present in all the spring varieties under investigation in its recessive state. Conclusions. Out of three dominant alleles in the studied sampling, the highest incidence was noted for allele Ppd-D1a (28.5 %). All the varieties from Mexico, present in the set, carry this allele. At the same time, it was not found in any variety from Russia. Allele Ppd-B1a was detected in the varieties from all the regions with the incidence of 7.7 (Russia) – 44.4 % (Asia). Allele Ppd-B1c was sporadically present in the varieties from Russia, Ukraine, the USA, Japan, and Brazil, and its incidence in the total sampling was insignificant (5.8 %). The varieties, identified by the allelic status of Ppd-1 genes, may be used as donors for selection and determination of the influence of alleles for each gene by the development rate and related economically valuable traits of bread wheat.

Phenology and Floret Development as Affected by the Interaction between Eps-7D and Ppd-D1

Earliness per se (Eps) genes may play a critical role in further improving wheat adaptation and fine-tuning wheat development to cope with climate change. There are only few studies on the detailed effect of Eps on wheat development and fewer on the interaction of Eps with the environment and other genes determining time to anthesis. Furthermore, it seems relevant to study every newly discovered Eps gene and its probable interactions as the mechanisms and detailed effects of each Eps may be quite different. In the present study, we evaluated NILs differing in the recently identified Eps-7D as well as in Ppd-D1 at three temperature regimes (9, 15 and 18 °C) under short day. The effect of Eps-7D on time to anthesis as well as on its component phases varied both qualitatively and quantitatively depending on the allelic status of Ppd-D1 and temperature, being larger in a photoperiod-sensitive background. A more noticeable effect of Eps-7D (when combined with Ppd-D1b) was realised during the late reproductive phase. Consequently, the final leaf number was not clearly altered by Eps-7D, while floret development of the labile florets (florets 2 and 3 in this case, depending on the particular spikelet) was favoured by the action of the Eps-7D-late allele, increasing the likelihood of particular florets to become fertile, and consequently, improving spike fertility when combined with Ppd-D1b.

Differential activity of PARP inhibitors in BRCA1- versus BRCA2-altered metastatic castration-resistant prostate cancer (mCRPC).

100 Background: Two PARP inhibitors (olaparib, rucaparib) are now FDA approved for mCRPC patients with mutations in BRCA1/2, but the relative efficacy of PARP inhibition in BRCA1- vs BRCA2-altered mCRPC is understudied. Methods: We conducted a multi-center retrospective analysis involving 11 academic sites. We collected genomic and clinical data from 123 BRCA1/2-altered mCRPC patients receiving PARP inhibitor treatment. The primary efficacy endpoint was PSA50 response rate (≥50% decline in PSA level). Secondary endpoints were PFS (clinical, radiographic or PSA progression, whichever occurred first) and overall survival (OS). We also captured information on other concurrent genomic alterations. We compared genomic characteristics and clinical outcomes among BRCA1- vs BRCA2-altered mCRPC. Results: A total of 123 patients (13 BRCA1, 110 BRCA2) were included. PARP inhibitors used were olaparib (n = 116), rucaparib (n = 3), talazoparib (n = 2) and veliparib (n = 2). All BRCA1 and 71% of BRCA2 patients had Gleason 8-10 disease. Compared to BRCA2 patients, BRCA1 patients were more likely to have received prior taxane chemotherapy (77% vs 62%). Age, baseline PSA, and prior enzalutamide/abiraterone treatment were similar between groups. PSA50 responses in BRCA1- vs BRCA2-altered patients were 38% vs 65% respectively ( P= 0.06). Median PFS in BRCA1 vs BRCA2 patients was 3.0 mo (95%CI, 0.9–5.1) vs 8.0 mo (95%CI, 5.3–10.6) respectively (HR 0.42, P= 0.01). Similarly, median OS in BRCA1 vs BRCA2 patients was 11.0 mo (95%CI, 9.9–12.1) vs 24.0 mo (95%CI, 18.2–29.8) respectively (HR 0.33, P= 0.005). There were roughly equal proportions of germline mutations (50% vs 45%) and biallelic mutations (31% vs 25%) in the BRCA2 and BRCA1 groups, respectively. There were numerically more TP53 mutations in the BRCA1 vs BRCA2 group (40% vs 29%, P= 0.45), but an equal number of mutations in other key genes ( PTEN, RB1 and AR). Conclusions: PARP inhibitor activity is diminished in BRCA1- vs BRCA2-altered mCRPC. In our cohort, this differential activity was not explained by mutation origin (germline vs somatic) or allelic status (mono- vs biallelic). These findings warrant validation.

Interactions between two QTLs for time to anthesis on spike development and fertility in wheat

Earliness per se (Eps) genes are reported to be important in fine-tuning flowering time in wheat independently of photoperiod (Ppd) and vernalisation (Vrn). Unlike Ppd and Vrn genes, Eps have relatively small effects and their physiological effect along with chromosomal position are not well defined. We evaluated eight lines derived from crossing two vernalisation insensitive lines, Paragon and Baj (late and early flowering respectively), to study the detailed effects of two newly identified QTLs, Eps-7D and Eps-2B and their interactions under field conditions. The effect of both QTLs was minor and was affected by the allelic status of the other. While the magnitude of effect of these QTLs on anthesis was similar, they are associated with very different profiles of pre-anthesis development which also depends on their interaction. Eps-7D affected both duration before and after terminal spikelet while not affecting final leaf number (FLN) so Eps-7D-early had a faster rate of leaf appearance. Eps-2B acted more specifically in the early reproductive phase and slightly altered FLN without affecting the leaf appearance rate. Both QTLs affected the spike fertility by altering the rate of floret development and mortality. The effect of Eps-2B was very small but consistent in that -late allele tended to produce more fertile florets.

Search for donors of powdery mildew resistance genes among seedless and table grape varieties

Powdery mildew (Erysiphe necator) is one of the most common and economically significant diseases of grapes. Currently, the main method of controlling the disease is pesticide treatment. Breeding of resistant varieties is necessary to reduce chemical treatments. Currently, a number of grape resistance genes to powdery mildew and DNA markers for identification the allelic status of these genes are known. In a study to determine the presence of resistance loci Ren3 and Ren9, 25 genotypes of table grape varieties were analyzed, including 18 seedless varieties. DNA markers GF15-42, ScORGF15-02 were used to identify Ren3 gene, and CenGen6 – to identify Ren9 gene. DNA of cultivars Regent and Seyve Villard 12-375, which have resistance alleles, were used as positive controls. As a result of DNA marker analysis, it was determined that genotypes of table varieties Viking, Kodryanka, Moldova, Nadezhda AZOS, Original and seedless varieties Pamyati Smirnova, Kishmish Zaporozhskiy and Kishmish 342 carry loci of resistance to powdery mildew Ren3 and Ren9.

Interactions between two QTLs for time to anthesis on spike development and fertility in wheat

Earliness per se (Eps) genes are reported to be important in fine-tuning flowering time in wheat independently of photoperiod (Ppd) and vernalisation (Vrn). Unlike Ppd and Vrn genes, Eps have relatively small effects and their physiological effect along with chromosomal position are not well defined. We evaluated eight lines derived from crossing Paragon and Baj (late and early flowering respectively), vernalisation insensitive, to study the detailed effects of two newly identified QTLs, Eps-7D and Eps-2B and their interactions under field conditions. The effect of both QTLs were minor but their effect was modulated by the allelic status of the other. While the magnitude of effect of these QTLs on anthesis was similar, they are associated with very different profiles of pre-anthesis development which also depends on their interaction. Eps-7D affected both duration before and after terminal spikelet while not affecting final leaf number (FLN) so Eps-7D-early had a faster rate of leaf appearance. Eps-2B acted more specifically in the early reproductive phase and slightly altered FLN without affecting the leaf appearance rate. Both Eps-7D and 2B affected the spike fertility by altering the rate of floret development and mortality. The effect of the latter was very small but consistent in that the -late allele tended to produced more fertile florets.

Marker-assisted selection for the gene of β-carotene hydroxylase in maize

Aim. Estimation of the allelic status of marker crtRB1-3'TE of the β-carotene hydroxylase gene and marker-assisted selection by this marker in Ukrainian maize breeding material. Methods. Field method and polymerase chain reaction. Results. The analysis of the allelic state of β-carotene hydroxylase gene for marker crtRB1-3'TE in maize breeding populations (DK23×F2)F2 and (DK23×F2)F3MAS having been obtained after the first and second self-pollinations of single cross DK23×F2 was provided. It was established that the parental inbred lines DK23 and F2 contained respectively 296 bp (unfavorable) and 543 bp (favorable) alleles of marker crtRB1-3'TE. The three kinds of genotypes appeared to present at different frequencies in (DK23×F2)F2 – homozygous for allele 296 bp, homozygous for allele 543 bp and heterozygous with both alleles 296 bp and 543 bp. For further cultivation and self-pollination, only plants with allele 543 bp within (DK23×F2)F2 were selected. All tested plants in population (DK23×F2)F3MAS were homozygous for allele 543 bp. Conclusions. Marker-associated selection in two generations for the β-carotene hydroxylase gene, involved in β-carotene accumulation, allowed to select homozygous plants of maize by favorable crtRB1-3'TE allele. Keywords: Zea mays L., molecular genetic markers, carotenoids, breeding populations, allele.

On the origin of photoperiod non-responsiveness in barley

In barley, the transition from the vegetative to reproductive phase is complex and under the control of photoperiodic and temperature conditions. One major gene involved is PPD-H1, a PSEUDO-RESPONSE REGULATOR 7 (PRR7) that encodes a component of the circadian clock. Mutation at PPD-H1 resulted in the photoperiod non-responsive ppd-H1 alleles that are beneficial under high latitudinal environments as they allow vegetative growth during the long-day summer conditions whereby higher yields are harvested by farmers. Utilizing a diverse GWAS panel of world-wide origin and a genome-wide gene-based set of 50K SNP markers, a strong association of days to heading with the PPD-H1 gene was detected in multi-location field trials. Re-sequencing of the gene spanning putative causative SNPs, SNP22 (Turner et al. 2005) and SNP48 (Jones et al. 2008), detected recombination between the two, previously reported to be in complete LD. Phenotyping of the recombinants and phylogenetic relationships among haplotypes supported the original conclusion of Turner et al. (2005) that SNP22, present in the CCT domain, is the most likely causative SNP. To infer the origin of non-responsiveness, the PPD-H1 gene was re-sequenced in a geo-referenced collection of 2057 wild and domesticated barleys and compared with the allelic status of the 6000-year-old barley sample from the Yoram cave in the Masada cliff. A monophyletic and post-domestication origin in the Fertile Crescent was found in contrast to the pre-domestication origin proposed by Jones et al. (2008). We show that the photoperiod non-responsiveness originated from Desert type wild barley in the Southern Levant.