Mean Allele Frequency Research Articles

Clonal haemopoiesis and therapy-related myeloid malignancies in elderly patients: a proof-of-concept, case-control study

Clonal haemopoiesis of indeterminate potential (CHIP) is an age-associated genetic event linked to increased risk of primary haematological malignancies and increased all-cause mortality, but the prevalence of CHIP in patients who develop therapy-related myeloid neoplasms is unknown. We did this study to investigate whether chemotherapy-treated patients with cancer who have CHIP are at increased risk of developing therapy-related myeloid neoplasms. We did a nested, case-control, proof-of-concept study to compare the prevalence of CHIP between patients with cancer who later developed therapy-related myeloid neoplasms (cases) and patients who did not develop these neoplasms (controls). We identified cases from our internal biorepository of 123 357 patients who consented to participate in the Total Cancer Care biobanking protocol at Moffitt Cancer Center (Tampa, FL, USA) between Jan 1, 2006, and June 1, 2016. We included all individuals who were diagnosed with a primary malignancy, were treated with chemotherapy, subsequently developed a therapy-related myeloid neoplasm, and were 70 years or older at either diagnosis. For inclusion in this study, individuals must have had a peripheral blood or mononuclear cell sample collected before the diagnosis of therapy-related myeloid neoplasm. Controls were individuals who were diagnosed with a primary malignancy at age 70 years or older and were treated with chemotherapy but did not develop therapy-related myeloid neoplasms. Controls were matched to cases in at least a 4:1 ratio on the basis of sex, primary tumour type, age at diagnosis, smoking status, chemotherapy drug class, and duration of follow-up. We used sequential targeted and whole-exome sequencing and described clonal evolution in cases for whom paired CHIP and therapy-related myeloid neoplasm samples were available. The primary endpoint of this study was the development of therapy-related myeloid neoplasm and the primary exposure was CHIP. We identified 13 cases and 56 case-matched controls. The prevalence of CHIP in all patients (23 [33%] of 69 patients) was higher than has previously been reported in elderly individuals without cancer (about 10%). Cases had a significantly higher prevalence of CHIP than did matched controls (eight [62%] of 13 cases vs 15 [27%] of 56 controls, p=0·024; odds ratio 5·75, 95% CI 1·52-25·09, p=0·013). The most commonly mutated genes in cases with CHIP were TET2 (three [38%] of eight patients) and TP53(three [38%] of eight patients), whereas controls most often had TET2 mutations (six [40%] of 15 patients). In most (four [67%] of six patients) cases for whom paired CHIP and therapy-related myeloid neoplasm samples were available, the mean allele frequency of CHIP mutations had expanded by the time of the therapy-related myeloid neoplasm diagnosis. However, a subset of paired samples (two [33%] of six patients) had CHIP mutations that decreased in allele frequency, giving way to expansion of a distinct mutant clone. Patients with cancer who have CHIP are at increased risk of developing therapy-related myeloid neoplasms. The distribution of CHIP-related gene mutations differs between individuals with therapy-related myeloid neoplasm and those without, suggesting that mutation-specific differences might exist in therapy-related myeloid neoplasm risk. Moffitt Cancer Center.

Clonal Hematopoiesis Is Associated with Therapy-Related Myeloid Malignancies in the Elderly

▪BackgroundClonal hematopoiesis of indeterminate potential (CHIP) is an age-dependent genetic event occurring in as high as 10% of individuals over age 70. Although the clinical consequence of CHIP is not well understood, it is linked to an increased risk of primary hematologic malignancies and increased all-cause mortality. A recent study reported a higher prevalence (26%) of CHIP in patients with advanced cancers (Zehir A, ASCO abstract, 2016). However, the prevalence of CHIP in patients developing therapy-related myeloid neoplasms (T-MN) is unknown. We hypothesize that chemotherapy treated cancer patients with CHIP are at higher risk for T-MN development. To address this, we conducted a nested case-control study to compare the prevalence of CHIP in cancer patients who later developed T-MN compared to those that did not. We performed sequential targeted and whole exome sequencing of T-MN cases and described clonal evolution in cases for who paired CHIP and T-MN samples were available.MethodsWe identified all patients who consented to our institutional biobanking protocol with a primary cancer preceding T-MN and an age ≥ 70 years at the time of either cancer. Chart reviews were conducted to verify that only patients who received chemotherapy and provided a blood sample before development of T-MN were included. Controls were defined as cancer patients who did not develop a subsequent malignancy and were matched to cases 4:1 on gender, primary tumor diagnosis, age, smoking status (ever/never), chemotherapy drug class, and follow-up time.DNA was isolated from peripheral blood (CHIP) or bone marrow samples (T-MN), and targeted amplicon based next-generation sequencing was performed to identify somatic mutations in genes associated with CHIP (49 genes, ThunderBolts Myeloid Panel, RainDance). Mesenchymal stem cells and CD3+ cells derived from the T-MN sample were used as independent germline controls to confirm somatic CHIP mutations. Whole exome sequencing was also performed (Agilent SureSelect XT Clinical Research Exome, Illumina NextSeq Sequencer) in paired CHIP and T-MN samples and clonal architecture was inferred using SciClone (Miller CA, PLoS Comput Biol, 2014). CHIP was defined in any sample that harbored a somatic mutation at an allele frequency of >3%.ResultsAfter screening 105,998 consented individuals from 2006 to 2016, a total of 13 patients (Figure 1a) were identified. The most common primary cancer in patients who developed T-MN was lymphoma, followed by adenocarcinoma (Table 1). The mean time to development of T-MN was 6.1 years and the mean time from the sampling date to the diagnosis of T-MN was 1.8 years. There were no statistical differences in the chemotherapy received by cases and controls (Table 1). The T-MN diagnosis was AML in 6 and MDS in 7 patients, and the majority (10/13) of patients harbored complex cytogenetics. T-MN patients had a poor overall survival (median 11 mo, Figure 1b). The prevalence of CHIP in cases and controls was higher (33%) than reported in individuals without cancer (10%), implying overlapping risk variables. Patients with T-MN had a significantly higher prevalence of CHIP prior to T-MN diagnosis when compared to matched controls (61.5% vs. 26.8%, p=0.02). The most commonly mutated gene in CHIP patients with T-MN was TP53 (36.4% vs. 5.4% in non-T-MN patients, p=0.02), while CHIP patients who did not develop T-MN more commonly harbored mutations in TET2 (35%) (Fig 1c). There was no difference in the mean allele frequencies (MAF) of CHIP mutations in cases vs. controls (9.4% vs. 14.3%, p=0.27). There was also no trend in the number or MAF of CHIP mutations observed and time to T-MN. In the majority of cases (83.3%), CHIP MAF expanded at the time of T-MN, however, a subset of paired samples (16.7%) harbored CHIP mutations that retracted giving way to expansion of a distinct mutant clone (Fig 1d).ConclusionsChip often precedes development of T-MN in chemotherapy-treated cancer patients. The distribution of CHIP-related gene mutations differs between individuals with T-MN vs. those without suggesting that there may be mutation-specific differences in T-MN risk. Further, although most CHIP mutations represented the founding leukemic mutation based on allelic frequency, some CHIP mutations retracted at the time of T-MN. These studies support the use of CHIP to inform individualized T-MN risk across cancer. [Display omitted] [Display omitted] DisclosuresKomrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau. Padron:Incyte: Research Funding; CTI: Research Funding; KALOBIOS: Research Funding.

Whole Genome Amplification and Exome Sequencing at the Single Cell Level - a Way to Address Clonal Heterogeneity and Very Sparse Clinical Material

Simonsen & al 2016Detailed characterization of sparse cancer subpopulations and single cells by next generation sequencing (NGS) modalities is rapidly gaining momentum, possibly yielding valuable information for personalized therapy. On the other hand, exome sequencing generally displays low coverage compared to targeted panels, target bias and broader allele frequency spectrums in contrast to whole genome sequencing (WGS). While single cell resolution is the ultimately feat for characterization of discrete subclonal contribution to leukemogenesis it is clear that allelic dropout generally occurs with high frequency, when performing whole genome amplification (WGA) and sequencing of single cells.Here, we hypothesized that detailed genomic profiling can be resolved from a minimal number of leukemic cells following WGA. We aimed to determine the cell number threshold by conventional laboratory analyses, and to elucidate what resolution to expect from whole exome sequencing (WES) at relatively low coverage.Methods6 single cells from the OCI-AML3 cell line were extracted manually by micromanipulation under a dissection microscope using a fine glass pipette, along with triplets of 2, 5 and 10 cells for microsatellite genotyping, along with serial dilutions to approximately 25 and 50 cells. Analysis encompassed 21 short tandem repeat loci, of which 18 are heterozygous in this cell line. Also, fragment analysis of NPM1W288fs mutation and qPCR of the DNMT3AR882Cmutation were performed after WGA (REPLI-g Single Cell Kit, Qiagen, Hilden, DE).For sequencing micromanipulated 5-cell assays were manually prepared, including samples with 25 and 50 cells as described above. Doublet samples underwent WGA and WES (Aros Applied, Eurofins, Aarhus, DK). Library preparation was performed by the vendor using Nextera Rapid Capture 37Mb kit (Illumina, San Diego, CA, USA). The six samples were whole exome sequenced aiming at a theoretical depth of 90x and data processed in parallel with Genome Analysis ToolKit (GATK 3.6, Broad institute, Cambridge, MA, USA) and CLC Biomedical Workbench 2 (Qiagen, Aarhus, DK). Copy number variations were resolved by chromosomal variant allele frequency (read depth threshold > 39) assessment and kernel density estimation.ResultsDNA yield from the WGA was approximately 35μg. NPM1 and DNMT3A mutations were detected by fragment analysis and qPCR, respectively. Microsatellite genotyping of single cells showed recurrent allele dropout compared to 2, 5 and 10 cells etc. The positive correlation between quality and cell number using microsatellite genotyping is shown in Fig. 1A. Detection of DNMT3AR882Cmutation was consistent from 2 cells and up.A mean of 9.4x107 reads was achieved (8.3-12.7x107) from exome sequencing with 99.3% of the sequences mapped to human reference genome GRCh37. NPM1 type A somatic mutation with insertion of tetranucleotide TCTG, leading to known somatic frameshift p.W288fs and somatic DNMT3AR882C, were detected in all sequenced subsets with 16 of 44 to 19 of 43 reads, respectively, and mean allele frequency of 41.5% (25.9-75%) and 44.5% (31.3-57.1%). All OCI-AML3 cell line copy number variations (CNVs) were confirmed (Table 1), although very noisy distributions were observed in samples with 5 cells (Fig. 1B-D). Additional lesions were also found, possibly acquired in culture or revealing inadequate characterization of the cell line, adding NRASQ61L, +7q, del(1p) and del(19q). As the latter deletions have not been described by conventional cytogenetics, these may be copy-neutral loss of heterozygosity (LoH).Discussion & conclusionThis study fills a gap in evaluating the feasibility of detecting somatic mutation and CNVs from extremely small amount of biological material, quantitatively and qualitatively. By choosing a very low number of cells from a well-characterized leukemia cell line we have simulated genomic profiling of a small subpopulation contributing to a more detailed picture of the cell biology. In perspective, this approach is applicable in direct combination with flow cytometry and for minimal residual disease characterization. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Genomic Alterations on Outcomes in Myelofibrosis Patients Undergoing JAK1/2 Inhibitor Therapy

Introduction:The advent of next generation sequencing (NGS) has brought intense interest to the complex genetic landscape of myeloproliferative neoplasms (MPN). However, data regarding clinical outcomes in the context of novel MPN therapies such as JAK inhibitors are scarce. Limited data indicate that high molecular risk signature (HMR, presence of at least one mutation in ASXL1, EZH2, IDH1/2, SRSF2) or multiple mutations may be associated with decreased spleen response and a shorter time to discontinuation of Ruxolitinib in myelofibrosis (Patel et al, Blood 2015).Methods:All myelofibrosis patients seen in the MPN program at Princess Margaret Hospital between November 2009 and May 2016 and treated with JAK1/2 inhibitor therapy were identified. NGS molecular profiling of 54 genes (39 hotspot region; 15 complete coding region coverage) was performed on peripheral blood or bone marrow samples using the TruSight Myeloid Sequencing Panel. Reporting was restricted to well-covered, exonic nonsynonymous, intronic splice site, and known pathogenic synonymous variants. Variants with global mean allele frequency >1% were identified using multiple population databases (1000 genomes, ESP, ExAC) and excluded. The primary endpoint was time from start of JAK1/2 inhibitor therapy to treatment failure (TTF) defined as treatment discontinuation, progression to accelerated phase or leukemic transformation, spleen progression or death. Secondary endpoints included best spleen, anemia and IWG response achieved by 48 weeks of treatment and overall survival. Response was assessed according to the 2013 revised IWG-MRT criteria. Transfusion dependency was assessed as any transfusion in 12 weeks prior to treatment or being identified as transfusion dependent in medical history.Results:Of 159 patients treated with JAK1/2 inhibitors at our institution, 102 met the inclusion criteria (see Table 1). Patients were excluded if; no sample was available for analysis (19), short use of JAK inhibitor prior to transplant (9), active clinical trial (5), in accelerated phase/acute leukemia (4) and others (20). First JAK inhibitor used was ruxolitinib in 77 patients and momelotinib in 25. At least one mutation was identified in every patient. Twenty (20%) patients had one mutation, 32 (31%) had 2 mutations and 50 (49%) patients had ≥ 3 mutations. Eighty (82%) patients had the JAK2V617F mutation, 15 (15%) had mutations in CALR, 4 (4%) had MPL mutations and one patient was triple negative. One patient had mutations in both CALR and JAK2 while another had mutations in MPL and CALR. Forty-eight (47%) patients had mutations consistent with HMR profile. Mutation profile is summarized in Table 2.With median follow-up of 2.5 years, 51 (50%) patients experienced treatment failure. On univariate analysis, TTF was associated with DIPSS, pre-treatment transfusion status and Hb <100 prior to initiating JAK inhibitor therapy. However, TTF was not associated with specific driver mutations, the number of mutations or HMR profile. Exploratory analysis of genes mutated in ≥5% of the population showed EZH2 (p=0.004) and CBL (p=0.005) mutated patients had shorter TTF. Multi-variable analysis employing anemia <100 and DIPSS with either of the number of mutations or HMR profile did not show any association with TTF. There was a trend towards TTF in patients with Hb<100 (HR 2.21, 95%CI: 0.99-4.95, p=0.05). Of 102 patients, 91 were evaluable for spleen response with 43 (47%) achieving a spleen response by 48 weeks of treatment. Twelve (23%) of the 53 patients evaluated met criteria for anemia response. Neither spleen nor anemia response was associated with the use of a particular JAK inhibitor. Of the 95 patients with at least 48-week follow-up, best response while on treatment was clinical improvement and spleen response in 29 (31%) and 19 (20%) patients respectively.Conclusions:In this study of myelofibrosis patients treated with JAK inhibitors, EZH2 and CBL mutated patients had shorter TTF. We did not find any association between TTF and number of mutations or other high risk mutations such as ASXL1/SRSF2. Anemia was the only significant independent predictor of shorter TTF. Our findings highlight the need for multicenter collaborative studies on a large number of patients and cautious use of mutation profiling results in routine clinical decision making with current treatment approaches.Spiegel and McNamara are co-primary authors. [Display omitted] [Display omitted] DisclosuresPanzarella:Cellgene: Consultancy. Schimmer:Novartis: Honoraria. Schuh:Amgen: Membership on an entity's Board of Directors or advisory committees. Yee:Novartis Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kamel-Reid:BMS: Research Funding. Gupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding.

The prevalence and distribution of the amyloidogenic transthyretin (TTR) V122I allele in Africa.

BackgroundTransthyretin (TTR) pV142I (rs76992529‐A) is one of the 113 variants in the human TTR gene associated with systemic amyloidosis. It results from a G to A transition at a CG dinucleotide in the codon for amino acid 122 of the mature protein (TTR V122I). The allele frequency is 0.0173 in African Americans.Methods PCR‐based assays to genotype 2767 DNA samples obtained from participants in genetic studies from various African populations supplemented with sequencing data from 529 samples within the 1000 Genomes Project.ResultsThe rs76992529‐A variant allele was most prevalent (allele frequency 0.0253) in the contiguous West African countries of Sierra Leone, Guinea, Ivory Coast, Burkina Faso, Ghana, and Nigeria. In other African countries, the mean allele frequency was 0.011.ConclusionsOur data are consistent with a small number of founder carriers of the amyloidogenic TTR V122I (p.Val142Ile) allele in southern West Africa, with no apparent advantage or disadvantage of an allele carrying newborn reaching adulthood. In U.S. African Americans, the allele represents a significant risk for congestive heart failure late in life. If clinical penetrance is similar in African countries with high allele frequencies, then cardiac amyloidosis could also represent a significant cause of heart disease in the elderly in those populations.

Expansion of the Knockdown Resistance Frequency Map for Human Head Lice (Phthiraptera: Pediculidae) in the United States Using Quantitative Sequencing.

Pediculosis is a prevalent parasitic infestation of humans, which is increasing due, in part, to the selection of lice resistant to either the pyrethrins or pyrethroid insecticides by the knockdown resistance (kdr) mechanism. To determine the extent and magnitude of the kdr-type mutations responsible for this resistance, lice were collected from 138 collection sites in 48 U.S. states from 22 July 2013 to 11 May 2015 and analyzed by quantitative sequencing. Previously published data were used for comparisons of the changes in the frequency of the kdr-type mutations over time. Mean percent resistance allele frequency (mean % RAF) values across the three mutation loci were determined from each collection site. The overall mean % RAF (±SD) for all analyzed lice was 98.3 ± 10%. 132/138 sites (95.6%) had a mean % RAF of 100%, five sites (3.7%) had intermediate values, and only a single site had no mutations (0.0%). Forty-two states (88%) had a mean % RAF of 100%. The frequencies of kdr-type mutations did not differ regardless of the human population size that the lice were collected from, indicating a uniformly high level of resistant alleles. The loss of efficacy of the Nix formulation (Prestige Brand, Tarrytown, NY) from 1998 to 2013 was correlated to the increase in kdr-type mutations. These data provide a plausible reason for the decrease in the effectiveness of permethrin in the Nix formulation, which is the parallel increase of kdr-type mutations in lice over time.

Evaluation of alternative management strategies for maintenance of genetic variation in wildlife populations

Wildlife management strategies are often designed around a population's demographic goals, but such strategies also can inadvertently impact genetic variation. For species like bison Bison bison, where management includes the regular removal of individuals to maintain restricted population sizes on constrained landscapes, management actions can be tailored to address genetic diversity retention in addition to simply maintaining a target population size. In this study, we provide an assessment of alternative culling strategies for maintenance of genetic variation in managed wildlife populations. Our primary goal was to compare the long-term retention of genetic variation and accumulation of inbreeding among three types of culling strategies, including one that considered genetic variation directly by measuring variation at a suite of variable loci [mean allele frequency (MAF) strategy], one that used genome-wide measures of variation [mean kinship (MK) strategy] and one that relied solely on demographic information (sex and age; RANDOM). To achieve this goal, we built an individual-based model, parameterized in accordance with bison biology, to project levels of genetic variation and inbreeding over time under each of the three management strategies. Our results suggest wildlife management strategies that incorporate goals for retaining genetic variation (MAF and MK strategies) are better suited to preserving the evolutionary potential of wildlife populations than those that focus solely on a target size and demographic stability (RANDOM). In particular, the MK culling strategy performed the best at maximizing the retention of genome-wide variation. These results extend previous work demonstrating the utility of pedigree-based mate selection strategies in captive population management, and show that such strategies maximize the retention of genome-wide variation under culling practices as well. These models will aid in the long-term management of bison, and can be adapted to other managed wildlife species.

Somatic Mutations Detected in Plasma Cell-Free DNA By Targeted Sequencing: Assessment of Liquid Biopsy in Primary Central Nervous System Lymphoma

BackgroundPrimary Central Nervous System Lymphoma (PCNSL) are rare and aggressive primary brain tumors. Histological diagnosis can be difficult at initial stages or at relapse due to deep brain structure involvement. Finding a minimally invasive biomarker aiding the diagnosis remains an unsolved question. Plasma Cell-Free DNA (cfDNA) seems to have shown its diagnostic and prognostic value in nodal Diffuse Large B Cell Lymphomas (DLBCL) [Roschewski et al, Lancet Oncology, 2015; Kurtz et al, Blood, 2015]. Our main objective was to demonstrate that targeted sequencing of cfDNA in plasma at time of diagnosis could identify PCNSL somatic mutations.Methods30 immuno-competent patients suffering from newly diagnosed PCNSL, without any extranevraxic lesions, were enrolled from 2008 to 2014. Tumor tissues and plasma samples were collected at the time of diagnosis and frozen until use. High throughput sequencing was performed on primitive tumors using a panel of 34 genes relevant to lymphomagenesis, as previously reported [Dubois et al, Oncotarget, 2015; Bohers et al, Haematologica, 2015]. We next performed patient-specific targeted sequencing of identified somatic mutations in cfDNA. The detection sensitivity threshold was set at 1% for all SNVs, except for MYD88 L265P, which was set at 0.1%. The primary endpoint was the proportion of patients having at least one somatic mutation found in the plasma.ResultsAmong 24 available plasmas, 15 patients (63%) had at least one detected somatic mutation in cfDNA. All plasmas had detectable cfDNA (mean concentration 1.6 ng/µL). No correlation was found between tumor volume and cfDNA concentration (R squared coefficient 0.01). Regarding the whole sequenced cohort (n=30) 21 (70%) were classified as nonGC subtype, 8 (27%) as GC subtype and 1 patient (3%) as unclassifiable, according to the Hans algorithm. 29 tumors had at least one somatic mutation, mainly nonsynonymous single nucleotide variants (SNV). The NF-kB pathway was the most affected by mutations: MYD88 (n=23, 77%), PIM1 (n=11, 37%), TNFAIP3 (n=6, 20%), IRF4 (n=3, 10%), CARD11 (n=3, 10%) and PRDM1 (n=3, 10%). Among the 23 tumors harboring a MYD88 mutation, the L265P variant was the most frequent (20 patients, 67%); mean tumor variant allele frequency was 46% [min 8%, max 91%]. One tumor harbored a single MYD88 L265P mutation with no other detectable abnormality. Among patients with both available plasma and a somatic MYD88 L265P mutation in the tumor, 15 patients (88%) had an identifiable L265P variant in cfDNA, with a mean variant allele frequency of 4% [min 0.1%, max 28%]. PIM1 and TNFAIP3 SNVs were also detected in cfDNA for respectively two and one patient.The second most affected pathway was the apoptotic pathway: genes affected by mutations included GNA13 (n=7, 23%), TP53 (n=2, 7%), MYC (n=1, 3%), CDKN2A (n=2, 7%) and BCL2 (n=1, 3%). The B Cell Receptor (BCR) pathway was also affected, mainly due to mutations targeting CD79B (n=10, 33%) and ITPKB (n=3, 10%) mutations. 8 tumors (27%) harbored a dual alteration affecting MYD88 and CD79B. One tumor of the GC subtype had one EZH2 SNV (Y646H), but the mutation was not found in cfDNA.There was no significant difference in overall survival (OS) between patients with and without mutations detected in cfDNA: mean OS 27 months versus 18 months (HR 0.6; IC95% [0.2 - 1.6], p value 0.3).Tumor gene copy number variations were detected in 29/30 patients with either homo or heterozygous deletions or copy gains. CDKN2A/2B deletions were detected in 23 cases (77%).ConclusionTo our knowledge this is the first study that provides evidence that somatic mutations can be detected in cfDNA in patients suffering from PCNSL, therefore constituting a minimally invasive tool helping for diagnosis. Further studies are now required to improve prognosis and predictive values of this new promising procedure for PCNSL patient care. DisclosuresNo relevant conflicts of interest to declare.

Serial Sequencing in Myelodysplastic Syndromes Reveals Dynamic Changes in Clonal Architecture and Allows for a New Prognostic Assessment of Mutations Detected in Cross-Sectional Testing

MDS and related disorders, including MDS/MPN and sAML that evolved from these conditions constitute disease continuum characterized by a wide spectrum of molecular lesions which often overlap. Here, we defined general mutational spectrum and clonal architecture in a large cohort (n=718) of MDS studied by whole exome sequencing (WES) and target deep sequencing. Within this cohort 97 cases were studied at multiple time points to clarify the clinical impact of clonal dynamics on phenotype commitment or outcomes. All samples were obtained after informed consent, according to protocols approved by the respective ethics boards of the participating institutions.When mean and maximum variant allele frequency (VAF) for whole mutations were at one time-point evaluated in disease phenotypes, significantly higher averaged values suggested their larger clones in sAML and CMML compared to MDS. Clustering analysis of multiple mutational events by Pyclone software discriminated the cases with multiple mutational clones (positive heterogeneity) and those with a single expansion of MDS clone (no heterogeneity detected). Over 80% of low-risk MDS and all the sAML harbored multiple clusters of mutations. These results suggest that intra-tumor heterogeneity of MDS is most likely due to various sizes of clonal and subclonal mutations, likely impacting clinical behavior.To delineate clonal dynamics in MDS, we assessed mutational burden and their temporal changes in serially collected samples (n=97). Among these, Pyclone analysis was applied to exome sequencing at two time points (n=11 pairs). All cases showed various mutational clusters with individual expansions and declines, including initially present, newly acquired or disappearing during clinical course. Initial subclones were identified at disease presentation in 55% of cases, of which in 86% the subclones expanded to occupy whole MDS population with clonal sweep. New subclones acquired during clinical course were identified in 91%, in which 60% cases harbored clonal sweep. Disappearing clones were observed in 55% of cases. Next, we applied clustering analysis on clonal size of driver mutations evaluated at multiple time points (n=97 cases) to categorize the most frequently mutated genes into 3 subtypes. Mutational burden of PTPN11 most frequently increased and were associated with leukemic evolution (an example of type I gene). Similarly, CBL, NRAS, STAG2, RUNX1, and IDH1 were categorized into the type I genes, demonstrating increased clonal size resulting in the evolutions into high-risk phenotypes. Although JAK2 mutations were related to the stable clinical course when the mutational burden decreased, cases with highly expanded JAK2 mutations resulted in leukemic evolution (occasional evolution or expansions; type II gene). DNMT3A, SRSF2, TP53, U2AF1, and ASXL1 mutations were also categorized into such type II consequences with occasional progression. The last category (type III) included clonal/founder genes EZH2, TET2, SF3B1 and PRPF8, demonstrating random shifts of clonal size and lack of association with leukemic evolution.The proposed hierarchical categorization correlates with clinical parameters. Cases with the increasing burden of type I gene mutations showed most significant increases in myeloblasts. Overall survival measured from second sampling time points in the cases with increasing type I mutations was significantly shorter in the whole cohort (HR=2.05, 95%CI; 1.14-3.79, P=0.016) and in the cases solely with IPSS INT-1 (HR=2.37, 95%CI; 1.01-5.97, P=0.048). Subcohorts classified according to the presence or absence of increasing type I mutations did not differ with regard to the IPSS categories. In contrast, increased mutational burden of type II and III genes did not correlated with any of the clinical parameters examined, even though some gene mutations including TP53, EZH2, and U2AF1 represented poor prognostic factors at disease presentation.In conclusion, this work demonstrates that detailed understanding of clonal dynamics allows for new insights into clinical significance of somatic mutations, made possible only by serial sample sequencing at multiple time points. Increasing clonal burden of extracted genes associated with predictive prognostic impact should be prospectively validated in more uniform and larger cohort of MDS. DisclosuresSekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Shih:Novartis: Research Funding.

Common Creatine Kinase gene mutation results in falsely reassuring CK levels in muscle disorders.

Learning point for clinicians The mean allele frequency of this SNP is 0.02 (∼2.5 million people in the UK are carriers). Therefore, greater awareness of this CKM SNP is needed as there may be instances where the diagnosis of rhabdomyolysis, statin-induced myopathy, poly-/dermatomyositis and other muscle conditions that result in raised CK’s, is missed in these patients. Creatine Kinase (CK) is the enzyme responsible for catalysing the exchange of phosphates in the creatine/phosphocreatine shuttle.1 Skeletal muscle CK is made …

Abstract 12295: Utilization of Whole-Exome Sequencing to Identify Causative Mutations in Familial Congenital Heart Disease

Congenital heart disease (CHD) is the most common type of birth defect, affecting ~8 out of every 1,000 live births, but there remains a limited understanding of the etiology for the majority of cases of CHD. Familial and population based studies have identified a strong genetic component to CHD, and familial cases of almost every type of CHD have been reported. The goal of this study was to identify pathogenic segregating variants in multiplex CHD families by whole-exome sequencing (WES). Target capture was performed using the Agilent SureSelectXT Target Enrichment kit, followed by WES on an Illumina HiSeq2500, on members from 9 families with Mendelian inherited CHD. Sequence alignment, post alignment processing, variant calling, and genotyping was performed with the Churchill pipeline. Tertiary analysis and annotation consisted of identifying rare (&lt;1% mean allele frequency in the population) segregating variants, prioritizing by matching to a CHD gene list, and predicting pathogenicity via 12 different utilities. This strategy utilized a list of 69 CHD genes selected under strict criteria and allowed for prioritization of the variants. Predicted pathogenic mutations were identified in 3 of the 9 families. A splice donor site mutation was identified in MYH11 (c.4599+1delC) in a family with autosomal dominant patent ductus arteriosus that is predicted to cause a 71 amino acid deletion, affecting functionality of the coiled-coil tail domain. A GATA4 mutation, p.G115W , was identified in a family with autosomal dominant atrial septal defects (ASD) and is predicted to affect the transactivation ability of GATA4 . A p.I263V mutation in TLL1 was identified in a family with autosomal dominant ASD; as this mutation was located in the astacin-like metalloprotease catalytic domain it is predicted to affect the enzymatic efficacy of TLL1 . In summary, our findings demonstrate the clinical utility of WES to successfully identify causative mutations in familial CHD and support the use of a CHD candidate gene list to allow for a more streamlined approach that enables rapid prioritization and identification of pathogenic variants from large WES data sets.

Spatial autocorrelation analysis and ecological niche modelling allows inference of range dynamics driving the population genetic structure of a Neotropical savanna tree

AbstractAimSpatial autocorrelation analysis of genetic diversity was combined with ecological niche modelling (ENM) to better infer how ecological and evolutionary processes underlie population structure in Eugenia dysenterica, a widely distributed tree in the ‘Cerrado’ region of Central Brazil.Location‘Cerrado’ region, Central Brazil.MethodsData were derived from 11 microsatellite loci in 23 populations of E. dysenterica, totalling 249 allele frequencies. The expected heterozygosity (He) within populations and the first principal coordinates extracted from pairwise FST and from the difference between RST and FST among populations were correlated with shifts in suitability from ENM. Frequencies were then analysed using a spatial autocorrelation analysis based on Moran's I and Mantel tests to contrast population differentiation for mean allele frequencies, allele size and shifts in suitability since the Last Glacial Maximum inferred from ENM.ResultsSpatial correlograms based on Moran's I and Mantel tests showed a linear decrease in autocorrelation with distance, which revealed north‐west–south‐east gradients in allele frequencies, genetic diversity and differences between RST and FST. These spatial patterns varied among loci and alleles, and the strongest spatial patterns were found for more common alleles with higher levels of differentiation among populations and for those correlated with shifts in ENM suitability.Main conclusionsCurrent genetic diversity and population structure in E. dysenterica can be explained by geographical range shifts associated with Quaternary climate dynamics, thus demonstrating the value of applying spatial analyses to study the ecological and evolutionary processes underlying differentiation even within populations possessing a continuous distribution.

Abstract P4-12-12: Next generation sequencing-based analysis of BRCA1 and BRCA2 genes: Applicability for fast diagnostics of large samples

Abstract Purpose BRCA1&amp;2 gene mutation test for hereditary breast ovarian syndrome (HBOC) has been conducted mostly by Sanger sequencing. Currently, the next generation sequencing (NGS) is rapidly incorporated to the fields of cancer research and clinical diagnostics. Here we evaluated NGS-based results of BRCA gene analysis and compared with the results of Sanger sequencing for future diagnostic applications. Methods The patients (n=100) who have genetic counseling and decided to perform BRCA test were included. All coding regions of BRCA1 and BRCA2 analyzed by both of Sanger sequencing and NGS using Access Array BRCA1, BRCA2 and TP53 kit (Fluidigm, USA). Access array kit was designed to multiplex 48 samples simultaneously for 184 amplicons and the average sequencing depth per base was 6,500X using MiSeq sequencer (Illumina, USA). We developed analysis pipelines to avoid false negative results and detect all pathogenic mutations. The reads were aligned to a reference genome (NCBI human genome assembly build 37) using the BWA-MEM, then all candidate variants called with minimum filtering parameter. Results Total of 765 variants were detected by NGS and among them 616 variants (frameshift:22, nonsense:8, splicing:3, missense:290, and synonymous variants:293) were identical with the results from Sanger sequencing. When we evaluated the results of Sanger sequencing as standard methods, the mean allele frequency showed difference as 41.7% and 12.0% for true positive heterozygous variants (616) and false positive variants (149), respectively. Conclusions There was no false negative of pathogenic mutations from NGS. The BRCA mutation detection using NGS represented potential applicability in clinical diagnosis. Citation Format: Sun-Young Kong, Eunhae Cho, Junnam Lee, Myong Cheol Lim, Jahyun Jang, Boyoung Park, Kyong-Ah Yoon, Young-Ho Kim, Eun Sook Lee. Next generation sequencing-based analysis of BRCA1 and BRCA2 genes: Applicability for fast diagnostics of large samples [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-12.

Next-Generation Sequencing (NGS)-Based Profiling of Idiopathic Cytopenia of Undetermined Significance (ICUS) Identifies a Subset of Patients with Genomic Similarities to Lower-Risk Myelodysplastic Syndrome (MDS)

IntroductionThe term ICUS has been used to describe patients with persistent unexplained cytopenia(s) who do not meet the minimal diagnostic criteria for MDS. While studies on ICUS have been few and small to date, it is clear that a subset of patients do progress to overt MDS or acute myeloid leukemia (AML), supporting the concept of an early or pre-phase of MDS (Wimazal, Leuk Res 2007; Hanson, Leuk Res 2009; Schroeder, Ann Oncol 2010). Through the use of NGS-based profiling, the majority of patients with MDS are now known to harbor one or more somatic mutations in driver genes, with RNA splicing and epigenetic pathways most commonly altered (Haferlach, Leukemia 2014). The aim of this study is to elucidate the genomic landscape of ICUS and to evaluate its potential relationship to lower-risk MDS.MethodsDNA exon sequences in SF3B1, SRSF2, U2AF1, ZRSR2, TET2, IDH1, IDH2, DNMT3A, EZH2, ASXL1, SETBP1, TP53, PHF6, RUNX1, ETV6, CBL1, NRAS, KIT, JAK2, MPL, and NPM1 were determined from the bone marrow aspirates of 250 patients with ICUS and 90 patients with lower-risk MDS (7 MDS with isolated del(5q), 7 MDS-unclassified, 12 refractory cytopenia with unilineage dysplasia, 27 refractory cytopenia with multilineage dysplasia, and 37 refractory anemia with ring sideroblasts) using a clinically validated and sensitive NGS assay on the Illumina MiSeq platform. The lower limit of detection of the assay was 5% with a minimum depth of coverage of 500x. Mutations were compared for concordance between duplicate samples and annotated using software that queried databases and published literature containing somatic mutations and germline variants.ResultsOne or more somatic mutations were detected in 33% (82/250) of patients with ICUS. Among this mutated subgroup (ICUS-MUT), the mean number of mutations was 1.8 per patient. TET2 mutation, postulated to be an early genetic event in the pathogenesis of myeloid neoplasms, was the most common mutation detected (38%). DNMT3A (20%), ASXL1 (18%), SRSF2 (15%), and ZRSR2 (11%) mutations were the next most common, with the remaining mutations each detected in <10% of patients. The two most common pathways involved were epigenetic (65%) and RNA splicing (18%). The mean allele frequency for mutations was 33% (range 6-99%), with 14% of mutations having an allele frequency of <10%. The most common type of mutation was missense (60%), followed by frameshift (17%), nonsense (16%), splicing (4%), and other (3%). While no significant difference was observed in the median hemoglobin, absolute neutrophil count, and platelet count between the ICUS-MUT and wild-type (ICUS-WT) subgroups, the ICUS-MUT subgroup was significantly older (78 vs. 69 years, P < 0.0001) and more commonly male (male:female ratio of 1.3:1 vs. 0.8:1, P= 0.0139).In lower-risk MDS, one or more somatic mutations were detected in 83% (75/90) of patients. The mean number of mutations was 1.8 per patient, identical to that observed in ICUS-MUT. SF3B1 mutation, which is highly associated with ring sideroblasts, was the most common mutation detected (47%). ASXL1 (16%), TET2 (14%), and DNMT3A (10%) mutations were the next most common, with the remaining mutations each detected in <10% of patients. The two most common pathways involved were RNA splicing (58%) and epigenetic (49%). The mean allele frequency for mutations was 32% (range 5-89%), with 6% of mutations having an allele frequency of <10%. The most common type of mutation was missense (66%), followed by frameshift (14%), nonsense (12%), splicing (7%), and other (1%). While no significant difference was observed in the median absolute neutrophil count, platelet count, age, and male:female ratio between lower-risk MDS and ICUS-MUT, lower-risk MDS patients were significantly more anemic (hemoglobin 9.7 vs. 10.5 g/dL, P= 0.0058).ConclusionsThrough the use of NGS-based profiling, one or more somatic mutations were detected in 33% of patients with ICUS. The ICUS-MUT subgroup displayed clinical parameters that were more similar to lower-risk MDS than the ICUS-WT subgroup. The ICUS-MUT subgroup was also similar to lower-risk MDS at the genomic level, with similar pathways involved, mean number of mutations, mean allele frequency, and type of mutations observed. Prior studies have shown that a subset of patients with ICUS do progress to overt MDS or AML. NGS-based profiling may be helpful in identifying those patients with potential early or pre-phase of MDS. DisclosuresKwok:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Reddy:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Lin:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Flamholz:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Yung:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Dabbas:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. McGinniss:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Nahas:Genoptix, Inc., a Novartis company: Employment, Equity Ownership. Kines:Genoptix, Inc., a Novartis company: Employment. Xu:Genoptix, Inc., a Novartis company: Employment, Equity Ownership.

The genetical theory of social behaviour

We survey the population genetic basis of social evolution, using a logically consistent set of arguments to cover a wide range of biological scenarios. We start by reconsidering Hamilton's (Hamilton 1964 J. Theoret. Biol. 7, 1-16 (doi:10.1016/0022-5193(64)90038-4)) results for selection on a social trait under the assumptions of additive gene action, weak selection and constant environment and demography. This yields a prediction for the direction of allele frequency change in terms of phenotypic costs and benefits and genealogical concepts of relatedness, which holds for any frequency of the trait in the population, and provides the foundation for further developments and extensions. We then allow for any type of gene interaction within and between individuals, strong selection and fluctuating environments and demography, which may depend on the evolving trait itself. We reach three conclusions pertaining to selection on social behaviours under broad conditions. (i) Selection can be understood by focusing on a one-generation change in mean allele frequency, a computation which underpins the utility of reproductive value weights; (ii) in large populations under the assumptions of additive gene action and weak selection, this change is of constant sign for any allele frequency and is predicted by a phenotypic selection gradient; (iii) under the assumptions of trait substitution sequences, such phenotypic selection gradients suffice to characterize long-term multi-dimensional stochastic evolution, with almost no knowledge about the genetic details underlying the coevolving traits. Having such simple results about the effect of selection regardless of population structure and type of social interactions can help to delineate the common features of distinct biological processes. Finally, we clarify some persistent divergences within social evolution theory, with respect to exactness, synergies, maximization, dynamic sufficiency and the role of genetic arguments.

DNMT3a Mutations Define a Pre-Leukemic Stem Cell Reservoir In Human Acute Myeloid Leukemia

Leukemia in humans arises from the multistep accumulation of mutations. However, the identity of the cell of origin, the nature of the first genetic lesion and the order of subsequent mutations remain poorly understood, as most cases of de novo acute myeloid leukemia (AML) are diagnosed without prior observation of a pre-leukemic clonal expansion. As part of studies to examine intra-tumoral genetic heterogeneity in AML, we carried out deep targeted sequencing (read depth 250×) of 101 commonly mutated leukemia genes on samples from 12 patients at diagnosis. Normal T-cells from each sample were expanded in vitro to provide a non-leukemic hematopoietic tissue for comparison. In 3 of 4 patients, we unexpectedly identified DNMT3a mutation not only in AML cells but also in T-cells at a low allele frequency (1-20%). Other genetic alterations such as NPM1 mutation (mutNPM1) were found only in AML cells and not in T-cells, ruling out contaminating AML cells as the source of the DNMT3a signal in cultured T-cells. To investigate the prevalence of T cell involvement, an additional 71 samples from AML patients at diagnosis were screened by Sanger sequencing for DNMT3a mutations. 17 of 71 AML samples (24%) carried R882 codon mutations (mutDNMT3a), and 15 of 17 (88%) also carried mutNPM1. Mutant allele frequency in freshly isolated T-cells was measured by droplet digital PCR (ddPCR). mutDNMT3a with no evidence of mutNPM1 was detected in T-cells of 12 of 17 patients (70.5%), suggesting that DNMT3a mutation occurs before NPM1 mutation in an ancestral stem/progenitor cell that gives rise to both T-cells and the dominant AML clone present at diagnosis. To directly determine whether phenotypic stem/progenitor cells that carried the mutDNMT3a allele could be identified within the non-leukemic hematopoietic compartment of AML blood and bone marrow samples, we undertook genetic analysis of highly-resolved phenotypically-defined normal stem, progenitor and mature lymphoid cell fractions from 10 patient samples. mutDNMT3a without detectable mutNPM1was present in stem cells and all downstream progenitors, with mean allele frequency among multipotent progenitor (MPP), multilymphoid progenitor (MLP) and common myeloid progenitor (CMP) of 31.7%. mutDNMT3a and mutNPM1 were found together only in granulocyte monocyte progenitor (GMP) and CD33+ blasts. Importantly, even for patients in whom mutDNMT3a was not detected in mature lymphoid populations, mutDNMT3a without mutNPM1 was found in MPP, MLP, CMP, providing strong evidence that mutDNMT3a precedes mutNPM1 during leukemogenesis. Analysis of diagnostic and remission samples revealed similar or higher proportion of cells with mutDNMT3a alone at remission compared to diagnosis. Xenotransplantation of cells from the diagnostic samples of 2 patients with mutDNMT3a and mutNPM1generated predominantly non-leukemic multilineage grafts (18 of 19 mice) with predominance of cells bearing mutDNMT3a without mutNPM1 (mean allele frequency 57%), confirming that mutDNMT3a was present in HSC. Kinetic analysis at 8 and 16 weeks revealed increasing mutDNMT3a allele frequency in multilineage xenografts over time, suggesting that mutDNMT3a confers a competitive growth advantage over non-mutated HSC. Collectively, our results are consistent with the clonal expansion in AML patients of mutDNMT3a HSC that survive chemotherapy. These cells may therefore represent a reservoir for further genomic progression leading to relapse. Our findings now offer the possibility of therapeutic intervention during remission to eliminate these surviving pre-leukemic clones and prevent relapse in a large proportion of AML patients carrying mutDNMT3a. As well, our work provides a framework for the identification of other early events in leukemogenesis and examination of how these changes disrupt normal HSC function and lead to leukemia. Disclosures:Wang:Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.

Empirical distributions of F(ST) from large-scale human polymorphism data.

Studies of the apportionment of human genetic variation have long established that most human variation is within population groups and that the additional variation between population groups is small but greatest when comparing different continental populations. These studies often used Wright’s F ST that apportions the standardized variance in allele frequencies within and between population groups. Because local adaptations increase population differentiation, high-F ST may be found at closely linked loci under selection and used to identify genes undergoing directional or heterotic selection. We re-examined these processes using HapMap data. We analyzed 3 million SNPs on 602 samples from eight worldwide populations and a consensus subset of 1 million SNPs found in all populations. We identified four major features of the data: First, a hierarchically F ST analysis showed that only a paucity (12%) of the total genetic variation is distributed between continental populations and even a lesser genetic variation (1%) is found between intra-continental populations. Second, the global F ST distribution closely follows an exponential distribution. Third, although the overall F ST distribution is similarly shaped (inverse J), F ST distributions varies markedly by allele frequency when divided into non-overlapping groups by allele frequency range. Because the mean allele frequency is a crude indicator of allele age, these distributions mark the time-dependent change in genetic differentiation. Finally, the change in mean-F ST of these groups is linear in allele frequency. These results suggest that investigating the extremes of the F ST distribution for each allele frequency group is more efficient for detecting selection. Consequently, we demonstrate that such extreme SNPs are more clustered along the chromosomes than expected from linkage disequilibrium for each allele frequency group. These genomic regions are therefore likely candidates for natural selection.

Allele frequency in loci which control coat colours in Hucul horse population

The objective of the study was to determine the frequency of alleles which produce coat colours in Hucul horse population in Poland. The breed is included in the Global Strategy for Management of Farm Animal Genetic Resources, hence its gene pool should remain in unaltered state. Huculs are bay, black, blue dun, yellow dun, tobiano, and chestnut. Grey and chestnut Huculs have always been undesirable. The material consisted of all 1022 matings which resulted in subpopulations recorded in Studbook volumes. The recessive allele frequency was estimated as the square root of recessive genotype frequencies in ASIP (A), MC1R (E), DUN (D), KIT (To region), and STX17 (G) loci. The frequency in A and E loci in total parental generation was also estimated in test matings. Genotype distribution in the population was anticipated according to gamete frequency in sires and dams. Small Wahlund effect, F&lt;sub&gt;ST&lt;/sub&gt;and &amp;chi;&lt;sup&gt;2&lt;/sup&gt; values for allele distributions show that division into subpopulations did not influence the population genetic structure significantly. Mean recessive allele frequency in A, E, D, To, and G loci amounted to 0.521, 0.115, 0.878, 0.929, and 0.997, respectively, and in A and E loci it was similar to that assessed in test matings. More bay horses and fewer D diluted horses appeared in offspring than expected. A, e, d, and To allele frequency showed a rising tendency. The genetic structure in Hucul population is not constant and does not comply strictly with the preservation aim. Bay, non-diluted, and tobiano horses are preferred. The linkage between MC1R and KIT loci can make the selection against e allele difficult. Breeders&amp;rsquo; preferences may lead to undesired changes in the allele frequency. To avoid such risk, it is recommended to select horses strictly complying with the rules included in the breeding programme and mate the horses randomly from this aspect. &amp;nbsp;

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples

BackgroundThe central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility.ResultsIn total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total).ConclusionsThe results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.

Analysis of Genetic Diversity and Population Structure of Buckwheat (Fagopyrum esculentum Moench) Landraces of Korea Using SSR Markers

Buckwheat (Fagopyrum esculentum Moench), one of the minor crops grown in Korea belonging to the Polygonaceae family, is an annual crop widely cultivated in Asia, Europe, and America and has a character of outcrossing and self-incompatibility. The objective of this study was to analyze the genetic variability, phylogenetic relationships and population structure of buckwheat landraces of Korea using SSR markers. Ten microsatellite markers have been detected from a total of 79 alleles among the 179 buckwheat accessions were collected from Korea. The number of allele per marker locus (N A ) ranged from 2 (GB-FE-001, GB-FE-043 and GB-FE-055) to 31 (GB-FE-035) with an average of 7.9 alleles. GB-FE-035 was the most polymorphic with the highest PIC value 0.93. Major allele frequencies (M AF ) for the 10 polymorphic loci varied from 0.12 to 0.97 with a mean allele frequency of 0.57. The expected heterozygosity (H E ) values ranged from 0.05 to 0.94 with an average of 0.53. The observed heterozygosity (H O ) ranged from 0.06 to 0.92 with an average of 0.42. The overall polymorphic information contents (PIC) values ranged from 0.05 to 0.93 with an average of 0.48. The landrace accessions of buckwheat used in the present study were not distinctly grouped according to geographic distribution. The study concludes that the results revealed genetic differentiation was low according to the geographic region because of outcrossing and self-incompatibility. We reported that our analyses on the genetic diversity of common buckwheat cultivars of Korea were performed by using of microsatellite markers.