Epidemiological trends and survival of oropharyngeal cancer in a high HPV-prevalent area: A Danish population-based study from 2000 to 2020.

Denmark, alongside other Scandinavian countries, the United States, Canada, and the United Kingdom, has high prevalence of human papillomavirus (HPV). Our oropharyngeal squamous cell carcinoma (OPSCC) database includes all diagnosed cases in Eastern Denmark during a period of more than two decades. We investigated the incidence, survival, and recurrence of patients with OPSCC with combined p16- and HPV testing covering a consecutive 21-year period. Age-adjusted incidence rate (AAIR) per 100,000, survival models, and Cox proportional-hazards model were employed. Two thousand eight hundred thirty-four patients were included (57.5% HPV positive (HPV+)/p16 positive (p16+), 33.7% HPV negative (HPV-)/p16 negative (p16-), 4% HPV+/p16-, and 4.8% HPV-/p16+). The AAIR for all patients increased from 1.8 to 5.1 per 100,000 from 2000 to 2020 linked to an increasing AAIR of HPV+/p16+ OPSCCs from 0.9 to 3.5 per 100,000 from 2000 to 2020. The AAIR for the HPV-/p16- OPSCCs decreased from 1.6 to 1.4 from 2017 to 2020. HPV+/p16+ OPSCCs had a higher 5-year overall survival (OS) of 79.2% compared to the other subgroups (HPV+/p16- OS: 50.4%; HPV-/p16+ OS: 49.4%; HPV-/p16- OS: 35.1%). The AAIR of the total OPSCC group increased from year 2000 to 2020, driven by a rise in the HPV+/p16+ group. A decreasing incidence rate was observed for the HPV-/p16- OPSCCs from 2017 to 2020. The OS for HPV+/p16+ OPSCCs was significantly higher compared to all other HPV/p16 subgroups. Therefore, we recommend testing for combined HPV and p16 status in patients with OPSCC when selecting patients for clinical trials, especially in case of de-escalating/escalating.

Cyclic hematopoiesis in a mixed-breed dog: case report and brief review.

An 8-wk-old, male, mixed-breed puppy was adopted from a rescue organization. From the time of adoption, the puppy suffered episodes of illness affecting various organ systems, which resolved with supportive therapy but relapsed once medical therapy was discontinued. Review of the hematologic data revealed cyclic fluctuations in circulating blood cells. Cyclicity was most prominent in neutrophils, with recurrent severe neutropenia. Neutropenic episodes lasted 5-6 d, with regular cycles of 11-14 d between nadir neutrophil counts. Genetic testing determined that the patient was homozygous mutant for the frameshift mutation in the adaptor protein complex 3 ß-subunit (AP3B1) gene, originally identified in gray collies with cyclic hematopoiesis (CH). Pedigree information was not available, but the patient's features were phenotypically distinct from those of collies. We describe here a case of the AP3B1 mutation in a mixed-breed dog that did not resemble a collie, undescribed previously, to our knowledge. Our findings indicate that the AP3B1 mutation and CH are present within the general canine population and are not restricted to collies.

The prevalence of tumour markers in malignant pleural effusions associated with primary pulmonary adenocarcinoma: a retrospective study.

BACKGROUND: Oncological treatment of primary pulmonary adenocarcinoma (AC) includes drugs targeting the pathways involving programmed death-ligand 1 (PD-L1), epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK). The aim of the study was to report the prevalence of these tumour markers in pleural fluid with cytology positive for pulmonary AC and the potential influence of volume pleural fluid tested. METHODS: We retrospectively reviewed all thoracenteses performed in a two-year period at our interventional unit at Department of Respiratory Medicine at Zealand University Hospital Naestved, Denmark. ALK and PD-L1 testing was done using immunohistochemistry and EGFR testing using next-generation sequencing. We included pleural fluid specimens containing malignant cells originating from primary pulmonary AC and with at least one tumour marker requested by the clinicians. RESULTS: When screening 927 pleural fluid specimens, we identified 57 in accordance with the inclusion criteria. PD-L1, ALK and EGFR were obtained in 35/55 (64%), 38/57 (67%) and 26/47 (55%), respectively. The prevalence did not increase when analysing volumes > 50 mL (p = 0.21-0.58). CONCLUSION: Tumour markers in pleural fluid specimens containing cells from pulmonary AC can be demonstrated in more than half of the cases. Therefore, supplementary invasive procedures than thoracentesis could potentially await these analyses.

Associations Between Cognitive Function and Levels of Glutamatergic Metabolites and Gamma-Aminobutyric Acid in Antipsychotic-Naïve Patients With Schizophrenia or Psychosis.

BACKGROUND: Abnormal glutamate and GABA (gamma-aminobutyric acid) levels have been found in the early phase of schizophrenia and may underlie cognitive deficits. However, the association between cognitive function and levels of glutamatergic metabolites and GABA has not been investigated in a large group of antipsychotic-naïve patients. METHODS: In total, 56 antipsychotic-naïve patients with schizophrenia or psychotic disorder and 51 healthy control subjects underwent magnetic resonance spectroscopy to measure glutamate, glutamate+glutamine (Glx), and GABA levels in dorsal anterior cingulate cortex (ACC) and glutamate and Glx levels in left thalamus. The cognitive domains of attention, working memory, and IQ were assessed. RESULTS: The whole group of antipsychotic-naïve patients had lower levels of GABA in dorsal ACC (p = .03), and the subgroup of patients with a schizophrenia diagnosis had higher glutamate levels in thalamus (p = .01), but Glx levels in dorsal ACC and thalamus did not differ between groups. Glx levels in dorsal ACC were positively associated with working memory (logarithmically transformed: b = -.016 [higher score indicates worse performance], p = .005) and attention (b = .056, p = .035) in both patients and healthy control subjects, although the association with attention did not survive adjustment for multiple comparisons. CONCLUSIONS: The findings suggest a positive association between glutamatergic metabolites and cognitive function that do not differ between patients and healthy control subjects. Moreover, our data indicate that decreased GABAergic levels in dorsal ACC are involved in schizophrenia and psychotic disorder, whereas increased glutamate levels in thalamus seem to be implicated in schizophrenia pathophysiology. The findings imply that first-episode patients with cognitive deficits may gain from glutamate-modulating compounds.

Circulating miRNAs as Biomarker in Cancer.

Deregulation of microRNA expression has been shown to play an important role in human malignancies. The identification of circulating-free miRNAs in biofluids a decade ago led to great enthusiasm and motivation to develop non-invasive tests based on the expression of these small non-coding RNAs. Herein, we review the progress within the field of research for identifying circulating miRNA cancer biomarkers and discuss the advantages and challenges associated with this. We also discuss the methodological and analytical variables, which may influence the final miRNA quantification and the importance of standardizing pre-analytical, analytical, and post-analytical processes in order to enable a successful translation of the results from basic research into the clinics.

miRNA profiling identifies deregulated miRNAs associated with osteosarcoma development and time to metastasis in two large cohorts.

Osteosarcoma (OS) is an aggressive bone tumor primarily affecting children and adolescents. The etiology of OS is not fully understood. Thus, there is a great need to obtain a better understanding of OS development and progression. Alterations in miRNA expression contribute to the required molecular alterations for neoplastic initiation and progression. This study is the first to investigate miRNA expression in OS in a large discovery and validation cohort comprising a total of 101 OS samples. We established the signature of altered miRNA expression in OS by profiling the expression level of 752 miRNAs in 23 OS samples using sensitive LNA-enhanced qPCR assays. The identified miRNA expression changes were correlated with gene expression in the same samples. Furthermore, miRNA expression changes were validated in a second independent cohort consisting of 78 OS samples. Analysis of 752 miRNAs in the discovery cohort led to the identification of 33 deregulated miRNAs in OS. Twenty-nine miRNAs were validated with statistical significance in the second cohort comprising 78 OS samples. miRNA/mRNA targets were determined, and 361 genes with an inverse expression of the target miRNA were identified. Both the miRNAs and the identified target genes were associated with multiple pathways related to cancer as well as bone cell biology, thereby correlating the deregulated miRNAs with OS tumorigenesis. An analysis of the prognostic value of the 29 miRNAs identified miR-221/miR-222 to be significantly associated with time to metastasis in both cohorts. This study contributes to a more profound understanding of OS tumorigenesis, by substantiating the importance of miRNA deregulation. We have identified and validated 29 deregulated miRNAs in the - to our knowledge - largest discovery and validation cohorts used so far for miRNA analyses in OS. Two of the miRNAs showed a promising potential as prognostic biomarkers for the aggressiveness of OS.

A Summary of the Biological Processes, Disease-Associated Changes, and Clinical Applications of DNA Methylation.

DNA methylation at cytosines followed by guanines, CpGs, forms one of the multiple layers of epigenetic mechanisms controlling and modulating gene expression through chromatin structure. It closely interacts with histone modifications and chromatin remodeling complexes to form the local genomic and higher-order chromatin landscape. DNA methylation is essential for proper mammalian development, crucial for imprinting and plays a role in maintaining genomic stability. DNA methylation patterns are susceptible to change in response to environmental stimuli such as diet or toxins, whereby the epigenome seems to be most vulnerable during early life. Changes of DNA methylation levels and patterns have been widely studied in several diseases, especially cancer, where interest has focused on biomarkers for early detection of cancer development, accurate diagnosis, and response to treatment, but have also been shown to occur in many other complex diseases. Recent advances in epigenome engineering technologies allow now for the large-scale assessment of the functional relevance of DNA methylation. As a stable nucleic acid-based modification that is technically easy to handle and which can be analyzed with great reproducibility and accuracy by different laboratories, DNA methylation is a promising biomarker for many applications.

Effects of milling on the extraction efficiency of incurred pesticides in cereals.

This study investigated the effects of particle size and milling temperature on the extraction efficiencies of pesticide residues from cereal flour. Samples of cereal grains (barley, oat, rye and wheat) were milled using a centrifugal mill with four different sieves (0.2, 1.0, 3.0 and 5.0 mm) or a knife mill both at room temperature and after freezing of the grain at -80°C overnight. The incurred pesticides in the test materials were extracted by the QuEChERS method and analysed by LC-MS/MS and GC-MS/MS. The particle size distribution for the milled samples was determined using a vibratory sieve shaker. Based on the pesticide levels recovered from each of the different millings and the corresponding particle size distributions, it was confirmed that smaller average particle sizes increase the extraction efficiency up to 31%, with all other factors equal. The cereals milled at room temperature produced lower pesticide extraction efficiencies compared with cereals milled when still frozen, especially for heat-sensitive pesticides. Furthermore, milling frozen grains was easier and resulted in more homogeneous samples with smaller relative particle sizes.

Stability study of methotrexate in 0.9% sodium chloride injection and 5% dextrose injection with limit tests for impurities.

PURPOSE: Results of an evaluation of the stability of methotrexate in 0.9% sodium chloride injection and 5% dextrose injection are presented. METHODS: Methotrexate concentrated solution (100 mg/mL) was diluted to nominal concentrations of 0.2 and 20 mg/mL in infusion bags containing 0.9% sodium chloride injection or 5% dextrose injection. The filled bags were stored for 28 days at 25 °C and 60% relative humidity and protected from light. Samples were withdrawn for analysis on the day of preparation and after 3, 7, 14, 21, and 28 days. The test program included visual inspections, measurements of pH and infusion bag weight loss, and high-performance liquid chromatography assays to determine methotrexate content and characterize degradation products. RESULTS: At both evaluated concentrations, methotrexate in 0.9% sodium chloride injection was stable for 28 days; only minor (<0.05%) increases in amounts of known and unknown degradation products were detected. In 5% dextrose injection, methotrexate at the higher concentration was stable for 28 days, with minor formation of degradation products; in the 0.2-mg/mL solution, however, methotrexate was stable for only 3 days. At later time points, an unknown impurity present at a concentration higher than 0.1% was observed. CONCLUSION: At concentrations of 0.2 and 20 mg/mL, methotrexate in 0.9% sodium chloride injection was found to be stable for 28 days when stored at 25 °C and protected from light. Under the same storage conditions, methotrexate in a 20-mg/mL solution prepared with 5% dextrose injection was stable for 28 days, whereas a 0.2-mg/mL solution in the same diluent was stable for only 3 days.

Tryptophan 2,3-dioxygenase (TDO)-reactive T cells differ in their functional characteristics in health and cancer.

Tryptophan-2,3-dioxygenase (TDO) physiologically regulates systemic tryptophan levels in the liver. However, numerous studies have linked cancer with activation of local and systemic tryptophan metabolism. Indeed, similar to other heme dioxygenases TDO is constitutively expressed in many cancers. In the present study, we detected the presence of both CD8+ and CD4+ T-cell reactivity toward TDO in peripheral blood of patients with malignant melanoma (MM) or breast cancer (BC) as well as healthy subjects. However, TDO-reactive CD4+ T cells constituted distinct functional phenotypes in health and disease. In healthy subjects these cells predominately comprised interferon (IFN)γ and tumor necrosis factor (TNF)-α producing Th1 cells, while in cancer patients TDO-reactive CD4+ T-cells were more differentiated with release of not only IFNγ and TNFα, but also interleukin (IL)-17 and IL-10 in response to TDO-derived MHC-class II restricted peptides. Hence, in healthy donors (HD) a Th1 helper response was predominant, whereas in cancer patients CD4+ T-cell responses were skewed toward a regulatory T cell (Treg) response. Furthermore, MM patients hosting a TDO-specific IL-17 response showed a trend toward an improved overall survival (OS) compared to MM patients with IL-10 producing, TDO-reactive CD4+ T cells. For further characterization, we isolated and expanded both CD8+ and CD4+ TDO-reactive T cells in vitro. TDO-reactive CD8+ T cells were able to kill HLA-matched tumor cells of different origin. Interestingly, the processed and presented TDO-derived epitopes varied between different cancer cells. With respect to CD4+ TDO-reactive T cells, in vitro expanded T-cell cultures comprised a Th1 and/or a Treg phenotype. In summary, our data demonstrate that the immune modulating enzyme TDO is a target for CD8+ and CD4+ T cell responses both in healthy subjects as well as patients with cancer; notably, however, the functional phenotype of these T-cell responses differ depending on the respective conditions of the host.

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA.

Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.

Effects of dopamine D2/D3 blockade on human sensory and sensorimotor gating in initially antipsychotic-naive, first-episode schizophrenia patients.

It has been suggested that psychophysiological measures of sensory and sensorimotor gating, P50 gating and prepulse inhibition of the startle reflex (PPI), underlie core features of schizophrenia and are linked to dopaminergic pathways in the striatum and prefrontal cortex. In the present study, the effects of a potent D2/D3 receptor antagonist, amisulpride, were investigated on PPI and P50 gating in a large sample of antipsychotic-naive, first-episode patients with schizophrenia. A total of 52 initially antipsychotic-naive, first-episode schizophrenia patients were assessed for their P50 gating, PPI, and habituation/sensitization abilities at baseline and after 2 and 6 weeks of treatment with flexible doses of amisulpride. In addition, 47 matched healthy controls were assessed at baseline and after 6 weeks. At baseline, the patients showed significantly reduced PPI, yet normal levels of P50 gating, habituation, and sensitization. Treatment with amisulpride showed no effects on these measures, either at 2 or 6 weeks of follow-up. This is the first study investigating the effects of monotherapy with a relatively selective dopamine D2/D3 receptor antagonist (amisulpride) on sensory and sensorimotor gating deficits in a longitudinal study of a large group of initially antipsychotic-naive, first-episode patients with schizophrenia. Our finding that amisulpride effectively reduced symptom severity in our patients without reducing their PPI deficits indicates that increased activity of dopamine D2 receptors may be involved in symptomatology of patients with schizophrenia, but not in their sensorimotor gating deficits.

The immune checkpoint regulator PD-L1 is a specific target for naturally occurring CD4+ T cells.

Programmed cell death 1 ligand 1 (PD-L1) is an important regulator of T-cell responses and may consequently limit anticancer immunity. We have recently identified PD-L1-specific, cytotoxic CD8+ T cells. In the present study, we develop these findings and report that CD4+ helper T cells spontaneously recognize PD-L1. We examined the locality of a previously identified HLA-A*0201-restricted PD-L1-epitope for the presence of possible CD4+ T-cell epitopes. Thus, we identified naturally occurring PD-L1-specific CD4+ T cells among the peripheral blood lymphocytes of cancer patients and - to lesser extents - healthy donors, by means of ELISPOT assays. PD-L1-specific CD4+ T cells appeared to be TH17 cells exhibiting an effector T-cell cytokine profile. Hence, PD-L1-specific CD4+ T cells released interferon γ (IFNγ), tumor necrosis factor α (TNFα) and interleukin-17 (IL-17) in response to a long PD-L1-derived peptide. Furthermore, we demonstrate that the specific recognition of PD-L1 by CD4+ T cells is MHC class II-restricted. Natural T-cell responses against PD-L1 are noteworthy as they may play a prominent role in the regulation of the immune system. Thus, cytokine release from PD-L1-specific CD4+ T cells may surmount the overall immunosuppressive actions of this immune checkpoint regulator. Moreover, PD-L1-specific T cells might be useful for anticancer immunotherapy, as they may counteract common mechanisms of immune escape mediated by the PD-L1/PD-1 pathway.

HLA-restricted CTL that are specific for the immune checkpoint ligand PD-L1 occur with high frequency in cancer patients.

PD-L1 (CD274) contributes to functional exhaustion of T cells and limits immune responses in patients with cancer. In this study, we report the identification of an human leukocyte antigen (HLA)-A2-restricted epitope from PD-L1, and we describe natural, cytolytic T-cell reactivity against PD-L1 in the peripheral blood of patients with cancer and healthy individuals. Notably, PD-L1-specific T cells were able not only to recognize and kill tumor cells but also PD-L1-expressing dendritic cells in a PD-L1-dependent manner, insofar as PD-L1 ablation rescued dendritic cells from killing. Furthermore, by incubating nonprofessional antigen-presenting cells with long peptides from PD-L1, we found that PD-L1 was rapidly internalized, processed, and cross-presented by HLA-A2 on the cell surface. Apparently, this cross-presentation was TAP-independent, as it was conducted not only by B cells but in addition by TAP-deficient T2-cells. This is intriguing, as soluble PD-L1 has been detected in the sera from patients with cancer. PD-L1-specific CTL may boost immunity by the killing of immunosuppressive tumor cells as well as regulatory cells. However, PD-L1-specific CTLs may as well suppress immunity by the elimination of normal immune cells especially PD-L1 expressing mature dendritic cells.

Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.

Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis.

Estimation of allele frequency and association mapping using next-generation sequencing data.

BACKGROUND: Estimation of allele frequency is of fundamental importance in population genetic analyses and in association mapping. In most studies using next-generation sequencing, a cost effective approach is to use medium or low-coverage data (e.g., < 15X). However, SNP calling and allele frequency estimation in such studies is associated with substantial statistical uncertainty because of varying coverage and high error rates. RESULTS: We evaluate a new maximum likelihood method for estimating allele frequencies in low and medium coverage next-generation sequencing data. The method is based on integrating over uncertainty in the data for each individual rather than first calling genotypes. This method can be applied to directly test for associations in case/control studies. We use simulations to compare the likelihood method to methods based on genotype calling, and show that the likelihood method outperforms the genotype calling methods in terms of: (1) accuracy of allele frequency estimation, (2) accuracy of the estimation of the distribution of allele frequencies across neutrally evolving sites, and (3) statistical power in association mapping studies. Using real re-sequencing data from 200 individuals obtained from an exon-capture experiment, we show that the patterns observed in the simulations are also found in real data. CONCLUSIONS: Overall, our results suggest that association mapping and estimation of allele frequencies should not be based on genotype calling in low to medium coverage data. Furthermore, if genotype calling methods are used, it is usually better not to filter genotypes based on the call confidence score.

Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variants.

Targeted capture combined with massively parallel exome sequencing is a promising approach to identify genetic variants implicated in human traits. We report exome sequencing of 200 individuals from Denmark with targeted capture of 18,654 coding genes and sequence coverage of each individual exome at an average depth of 12-fold. On average, about 95% of the target regions were covered by at least one read. We identified 121,870 SNPs in the sample population, including 53,081 coding SNPs (cSNPs). Using a statistical method for SNP calling and an estimation of allelic frequencies based on our population data, we derived the allele frequency spectrum of cSNPs with a minor allele frequency greater than 0.02. We identified a 1.8-fold excess of deleterious, non-syonomyous cSNPs over synonymous cSNPs in the low-frequency range (minor allele frequencies between 2% and 5%). This excess was more pronounced for X-linked SNPs, suggesting that deleterious substitutions are primarily recessive.

A genome-wide association study in the Japanese population identifies susceptibility loci for type 2 diabetes at UBE2E2 and C2CD4A-C2CD4B.

We conducted a genome-wide association study of type 2 diabetes (T2D) using 459,359 SNPs in a Japanese population with a three-stage study design (stage 1, 4,470 cases and 3,071 controls; stage 2, 2,886 cases and 3,087 controls; stage 3, 3,622 cases and 2,356 controls). We identified new associations in UBE2E2 on chromosome 3 and in C2CD4A-C2CD4B on chromosome 15 at genome-wide significant levels (rs7612463 in UBE2E2, combined P = 2.27 × 10⁻9; rs7172432 in C2CD4A-C2CD4B, combined P = 3.66 × 10⁻9). The association of these two loci with T2D was replicated in other east Asian populations. In the European populations, the C2CD4A-C2CD4B locus was significantly associated with T2D, and a combined analysis of all populations gave P = 8.78 × 10⁻¹4, whereas the UBE2E2 locus did not show association to T2D. In conclusion, we identified two new loci at UBE2E2 and C2CD4A-C2CD4B associated with susceptibility to T2D.

Studies of association between LPIN1 variants and common metabolic phenotypes among 17,538 Danes.

OBJECTIVE: Lipin-1, encoded by LPIN1, is expressed in the major metabolically active tissues. Decreased expression of lipin-1 in adipose tissue correlates with increased insulin resistance, and tagging of the LPIN1 locus has shown that rs33997857, rs6744682, and rs6708316 associate with metabolic phenotypes, specifically body mass index (BMI) and fasting serum lipid levels, both on the individual single-nucleotide polymorphism level and with a three-marker haplotype. Our aim was to validate the reported findings in the Danish population. DESIGN: In the present study, variants were analyzed in LPIN1 using case-control studies, haplotype analyses, and quantitative trait analyses in a population of 17,538 Danes. METHODS: The three LPIN1 variants were genotyped in 17,538 Danes from four study populations of middle-aged people. This provided us with a statistical power >99% to replicate previous findings. Variants were analyzed individually and in haplotype combinations in studies of quantitative metabolic traits and in case-control studies. RESULTS: None of the three variants were associated with the examined quantitative traits including BMI, waist circumference, blood pressure, fasting serum lipid concentrations, or plasma glucose or serum insulin concentrations in the fasting state and following an oral glucose tolerance test. Haplotypes were tested for association with quantitative traits; however, only nominal association with blood pressure (P=0.04) and waist circumference (P=0.04) was observed. In case-control studies, no association was found for individual variants or the three-marker haplotype. CONCLUSION: LPIN1 rs33997857, rs6744682, and rs6708316 did not associate with type 2 diabetes, obesity, or related quantitative metabolic phenotypes in the Danish population examined.