Classification of follicular-patterned thyroid lesions using a minimal set of epigenetic biomarkers.

Objective: The minimally invasive fine-needle aspiration cytology (FNAC) is the current gold standard for the diagnosis of thyroid nodule malignancy. However, the correct discrimination of follicular neoplasia often requires more invasive diagnostic techniques. The lack of suitable immunohistochemical markers to distinguish between follicular thyroid carcinoma and other types of follicular-derived lesions complicates diagnosis, and despite most of these tumours being surgically resected, only a small number will test positive for malignancy. As such, the development of new orthogonal diagnostic approaches may improve the accuracy of diagnosing thyroid nodules. Design: This study includes a retrospective, multi-centre training cohort including 54 fresh-frozen follicular-patterned thyroid samples and two independent, multi-centre validation cohorts of 103 snap-frozen biopsies and 33 FNAC samples, respectively. Methods: We performed a genome-wide genetic and epigenetic profiling of 54 fresh-frozen follicular-patterned thyroid samples using exome sequencing and the Illumina Human DNA Methylation EPIC platform. An extensive validation was performed using the bisulfite pyrosequencing technique. Results: Using a random forest approach, we developed a three-CpG marker-based diagnostic model that was subsequently validated using bisulfite pyrosequencing experiments. According to the validation cohort, this cost-effective method discriminates between benign and malignant nodules with a sensitivity and specificity of 97 and 88%, respectively (positive predictive value (PPV): 0.85, negative predictive value (NPV): 0.98). Conclusions: Our classification system based on a minimal set of epigenetic biomarkers can complement the potential of the diagnostic techniques currently available and would prioritize a considerable number of surgical interventions that are often performed due to uncertain cytology. Significance statement: In recent years, there has been a significant increase in the number of people diagnosed with thyroid nodules. The current challenge is their etiological diagnosis to discount malignancy without resorting to thyroidectomy. The method proposed here, based on DNA pyrosequencing assays, has high sensitivity (0.97) and specificity (0.88) for the identification of malignant thyroid nodules. This simple and cost-effective approach can complement expert pathologist evaluation to prioritize the classification of difficult-to-diagnose follicular-patterned thyroid lesions and track tumor evolution, including real-time monitoring of treatment efficacy, thereby stimulating adherence to health promotion programs.

Mortality risk in adults according to categories of impaired glucose metabolism after 18 years of follow-up in the North of Spain: The Asturias Study.

People who develop type 2 diabetes (T2D) are known to have a higher mortality risk. We estimated all-cause, cardiovascular, and cancer mortality-risks in our patient cohort according to categories of impaired glucose metabolism. This 18-year retrospective analysis included a region-wide, representative sample of a population aged 30-75 years. Age- and sex-stratified hazard ratios (HRs) were calculated for 48 participants with diagnosed T2D, 83 with undiagnosed T2D (HbA1c ≥6.5%, fasting glycemia ≥126 mg/dL, or glycemia after 75 g glucose load ≥200 mg/dL); 296 with prediabetes (HbA1c 5.7%-6.4%, fasting glycemia 100-125 mg/dL, or glycemia after 75 g glucose load 140-199 mg/dL), and 607 with normoglycemia. Over 18,612 person-years, 32 individuals with undiagnosed T2D, 30 with diagnosed T2D, 62 with prediabetes, and 80 with normoglycemia died. Total sample crude mortality rate (MR) was 10.96 deaths per 1,000 person-years of follow-up. MR of the diagnosed T2D group was more than 3-times higher and that of newly diagnosed T2D was 2-times higher (34.72 and 21.42, respectively) than total sample MR. Adjusted HR for all-cause mortality was 2.02 (95% confidence interval 1.29-3.16) and 1.57 (95% CI 1.00-2.28) in the diagnosed T2D group and the newly diagnosed T2D group, respectively. Adjusted HR for cardiovascular mortality in the T2D group was 2.79 (95% CI 1.35-5.75); this risk was greatly increased in women with T2D: 6.72 (95% CI 2.50-18.07). In Asturias, age- and sex-standardized all-cause mortality is more than 2-times higher for adults with T2D than for adults without T2D. The HR for cardiovascular mortality is considerably higher in T2D women than in normoglycemic women.

Epigenetic modulators of thyroid cancer.

There are some well known factors involved in the etiology of thyroid cancer, including iodine deficiency, radiation exposure at early ages, or some genetic changes. However, epigenetic modulators that may contribute to development of these tumors and be helpful to for both their diagnosis and treatment have recently been discovered. The currently known changes in DNA methylation, histone modifications, and non-coding RNAs in each type of thyroid carcinoma are reviewed here.

Epigenetic alterations in endocrine-related cancer.

Aberrant epigenetics is a hallmark of cancer, and endocrine-related tumors are no exception. Recent research has been identifying an ever-growing number of epigenetic alterations in both genomic DNA methylation and histone post-translational modification in tumors of the endocrine system. Novel microarray and ultra-deep sequencing technologies have allowed the identification of genome-wide epigenetic patterns in some tumor types such as adrenocortical, parathyroid, and breast carcinomas. However, in other cancer types, such as the multiple endocrine neoplasia syndromes and thyroid cancer, tumor information is limited to candidate genes alone. Future research should fill this gap and deepen our understanding of the functional role of these alterations in cancer, as well as defining their possible clinical uses.

DNA methylation signatures identify biologically distinct thyroid cancer subtypes.

OBJECTIVE: The purpose of this study was to determine the global patterns of aberrant DNA methylation in thyroid cancer. RESEARCH DESIGN AND METHODS: We have used DNA methylation arrays to determine, for the first time, the genome-wide promoter methylation status of papillary, follicular, medullary, and anaplastic thyroid tumors. RESULTS: We identified 262 and 352 hypermethylated and 13 and 21 hypomethylated genes in differentiated papillary and follicular tumors, respectively. Interestingly, the other tumor types analyzed displayed more hypomethylated genes (280 in anaplastic and 393 in medullary tumors) than aberrantly hypermethylated genes (86 in anaplastic and 131 in medullary tumors). Among the genes indentified, we show that 4 potential tumor suppressor genes (ADAMTS8, HOXB4, ZIC1, and KISS1R) and 4 potential oncogenes (INSL4, DPPA2, TCL1B, and NOTCH4) are frequently regulated by aberrant methylation in primary thyroid tumors. In addition, we show that aberrant promoter hypomethylation-associated overexpression of MAP17 might promote tumor growth in thyroid cancer. CONCLUSIONS: Thyroid cancer subtypes present differential promoter methylation signatures, and nondifferentiated subtypes are characterized by aberrant promoter hypomethylation rather than hypermethylation. Additional studies are needed to determine the potential clinical interest of the tumor subtype-specific DNA methylation signatures described herein and the role of aberrant promoter hypomethylation in nondifferentiated thyroid tumors.

Genetic and non-genetic predictors of LINE-1 methylation in leukocyte DNA.

BACKGROUND: Altered DNA methylation has been associated with various diseases. OBJECTIVE: We evaluated the association between levels of methylation in leukocyte DNA at long interspersed nuclear element 1 (LINE-1) and genetic and non-genetic characteristics of 892 control participants from the Spanish Bladder Cancer/EPICURO study. METHODS: We determined LINE-1 methylation levels by pyrosequencing. Individual data included demographics, smoking status, nutrient intake, toenail concentrations of 12 trace elements, xenobiotic metabolism gene variants, and 515 polymorphisms among 24 genes in the one-carbon metabolism pathway. To assess the association between LINE-1 methylation levels (percentage of methylated cytosines) and potential determinants, we estimated beta coefficients (ßs) by robust linear regression. RESULTS: Women had lower levels of LINE-1 methylation than men (ß = -0.7, p = 0.02). Persons who smoked blond tobacco showed lower methylation than nonsmokers (ß = -0.7, p = 0.03). Arsenic toenail concentration was inversely associated with LINE-1 methylation (ß = -3.6, p = 0.003). By contrast, iron (ß = 0.002, p = 0.009) and nickel (ß = 0.02, p = 0.004) were positively associated with LINE-1 methylation. Single nucleotide polymorphisms (SNPs) in DNMT3A (rs7581217-per allele, ß = 0.3, p = 0.002), TCN2 (rs9606756-GG, ß = 1.9, p = 0.008; rs4820887-AA, ß = 4.0, p = 4.8 × 10-7; rs9621049-TT, ß = 4.2, p = 4.7 × 10-9), AS3MT (rs7085104-GG, ß = 0.7, p = 0.001), SLC19A1 (rs914238, TC vs. TT: ß = 0.5 and CC vs. TT: ß = -0.3, global p = 0.0007) and MTHFS (rs1380642, CT vs. CC: ß = 0.3 and TT vs. CC; ß = -0.8, global p = 0.05) were associated with LINE-1 methylation. CONCLUSIONS: We identified several characteristics, environmental factors, and common genetic variants that predicted DNA methylation among study participants.

Epigenetic regulation of aging.

Aging is one of the most challenging and unresolved problems in biology owing to its highly complex nature. Public interest in aging has increased not only because all of us can expect to live to a ripe old age but also because we wish to avoid those age-related changes that lead to physical invalidity or other diseases (cancer, depression) and may ultimately cause social isolation. Aging is a process of genetic and epigenetic interactions at all biological levels, where epigenetics has an important function in determining the phenotypic differences that arise. Epigenetics also plays a key role in the development of diseases associated with aging and explains the relationship between an individual's genetic background, the environment, aging, and disease. DNA plasticity is mediated in part by the epigenetic changes that lead the role of a cell, and can be passed on to future generations. Epigenetics establishes the idea that our health can be affected not only by the interplay of our genes and environment but also by the inherited effects of our ancestors' genes and environment.

Transcriptional regulation of MICA and MICB: a novel polymorphism in MICB promoter alters transcriptional regulation by Sp1.

MHC class I-related genes A/B (MICA/B) are ligands of the NKG2D receptor expressed on T and NK cells. Their expression is highly restricted in normal tissues, but is up-regulated in tumoral and infected cells. We show that the minimal promoter of both genes contains a CCAAT box, which binds to NF-Y, and a GC box, which binds to Sp1, Sp3 and Sp4. We also demonstrate that MICB promoter is polymorphic, showing three single nucleotide polymorphisms (C>G at +16, -341, -408) and a deletion of two base pairs at -66 (AG>--) that is located close to the CCAAT box (-70) and the GC box (-86). Transcriptional activity associated with MICB promoter variants carrying this deletion, present in the 45.3% of Spanish population, showed a remarkable decrease (18-fold, p <0.01). By functional analysis, we show that the deletion plays a critical role in MICB promoter activity by diminishing Sp1 transcriptional activation. These important variations in MICB expression among normal individuals could imply a significant difference in the natural immune response against infections or tumor transformation, and might thereby contribute to an increased aberrant immune response against self cells, providing the molecular basis for the associations of the MICB gene to different autoimmune diseases.

MHC class I chain-related gene B promoter polymorphisms and celiac disease.

The possibility that susceptibility to celiac disease (CD) might be influenced by the MHC class I chain-related gene family, MICA and MICB, has been previously reported. In this study, we analyzed the MICB promoter and examined the association of the polymorphisms found within such in a group of CD patients. To study the MICB promoter we sequenced the 5' flanking region of MICB gene in DNA from homozygous B-lymphoblastoid cell lines corresponding to the most frequent MICB alleles found in our population (MICB*00502, MICB*002, MICB*004, and MICB*008). DNA from a MICB*003 homozygous individual was also analyzed. Sequence analysis revealed six single nucleotide polymorphisms located at positions 45860 C/A, 45862 G/C, 45877 C/G, 46113 A/C, 46219 G/C, and 46286 G/C and an insertion of 2 bp --/AG at position 45944 according to the published genomic sequence. Those polymorphisms were found to be associated in four different haplotypes corresponding to different MICB alleles. Subsequently, 126 CD subjects and 117 healthy controls were typed by polymerase chain reaction using sequence-specific primers for these polymorphisms. MICB promoter polymorphism haplotypes were also found in our population and showed strong linkage disequilibrium with MICB alleles. MICB promoter polymorphism Haplotype 3, included in MICB*002 and MICB*008 alleles, was found to be overrepresented in CD patients (79.4% CD patients vs 45.3% healthy controls; p(c) < 0.0001; OR = 4.64; CI 95% = 2.64-8.16). Both MICB*008 and MICB*002 alleles were found as part of the CD susceptibility extended haplotypes B8/DR3/DQ2, B18/DR3/DQ2, and DR4/DQ8.

Protective effect of the HLA-Bw4I80 epitope and the killer cell immunoglobulin-like receptor 3DS1 gene against the development of hepatocellular carcinoma in patients with hepatitis C virus infection.

The aim of the present study was to investigate, in 152 Spanish patients infected with hepatitis C virus (HCV), the possibility that killer cell immunoglobulin-like receptors (KIRs) influence progression to hepatocellular carcinoma. KIRs are related to the activation and inhibition of natural killer cells and may play an important role in the innate response against infection with such viruses as HCV. We found that the human leukocyte antigen-Bw4I80 epitope and the KIR3DS1 gene were more frequent in HCV carriers than in patients with hepatocellular carcinoma. Moreover, these associations were not independent of each other--the KIR3DS1/Bw4I80 genotype clearly was also more frequent in HCV carriers (odds ratio, 24.22).