Strategies for single base gene editing in an immortalized human cell line by CRISPR/Cas9 technology.

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03878-4.

A Drug Screening Revealed Novel Potential Agents against Malignant Pleural Mesothelioma.

The lack of effective therapies remains one of the main challenges for malignant pleural mesothelioma (MPM). In this perspective, drug repositioning could accelerate the identification of novel treatments. We screened 1170 FDA-approved drugs on a SV40-immortalized mesothelial (MeT-5A) and five MPM (Mero-14, Mero-25, IST-Mes2, NCI-H28 and MSTO-211H) cell lines. Biological assays were carried out for 41 drugs, showing the highest cytotoxicity and for whom there were a complete lack of published literature in MPM. Cytotoxicity and caspase activation were evaluated with commercially available kits and cell proliferation was assayed using MTT assay and by clonogenic activity with standard protocols. Moreover, the five most effective drugs were further evaluated on patient-derived primary MPM cell lines. The most active molecules were cephalomannine, ouabain, alexidine, thonzonium bromide, and emetine. Except for alexidine, these drugs inhibited the clonogenic ability and caspase activation in all cancer lines tested. The proliferation was inhibited also on an extended panel of cell lines, including primary MPM cells. Thus, we suggest that cephalomannine, ouabain, thonzonium bromide, and emetine could represent novel candidates to be repurposed for improving the arsenal of therapeutic weapons in the fight against MPM.

Pro64His (rs4644) Polymorphism Within Galectin-3 Is a Risk Factor of Differentiated Thyroid Carcinoma and Affects the Transcriptome of Thyrocytes Engineered via CRISPR/Cas9 System.

Background: Galectin-3 (LGALS3) is an important glycoprotein involved in the malignant transformation of thyrocytes acting in the extracellular matrix, cytoplasm, and nucleus where it regulates TTF-1 and TCF4 transcription factors. Within LGALS3 gene, a common single-nucleotide polymorphism (SNP) (c.191C>A, p.Pro64His; rs4644) encoding for the variant Proline to Histidine at codon 64 has been extensively studied. However, data on rs4644 in the context of thyroid cancer are lacking. Thus, the aim of the present work was to evaluate the role of the rs4644 SNP as risk factor for differentiated thyroid cancer (DTC) and to determine the effect on the transcriptome in thyrocytes. Methods: A case/control association study in 1223 controls and 1142 unrelated consecutive DTC patients was carried out to evaluate the association between rs4644-P64H and the risk of DTC. We used the nonmalignant cell line Nthy-Ori (rs4644-C/A) and the CRISPR/Cas9 technique to generate isogenic cells carrying either the rs4644-A/A or rs4644-C/C homozygosis. Then, the transcriptome of the derivative and unmodified parental cells was analyzed by RNA-seq. Genes differentially expressed were validated by quantitative reverse transcription PCR and further tested in the parental Nthy-Ori cells after LGALS3 gene silencing, to investigate whether the expression of target genes was dependent on galectin-3 levels. Results: rs4644 AA genotype was associated with a reduced risk of DTC (compared with CC, ORadj = 0.66; 95% confidence interval = 0.46-0.93; Pass = 0.02). We found that rs4644 affects galectin-3 as a transcriptional coregulator. Among 34 genes affected by rs4644, HES1, HSPA6, SPC24, and NHS were of particular interest since their expression was rs4644-dependent (CC>AA for the first and AA>CC for the others), also in 574 thyroid tissues of Genotype-Tissue Expression (GTEx) biobank. Moreover, the expression of these genes was regulated by LGALS3-silencing. Using the proximity ligation assay in Nthy-Ori cells, we found that the TTF-1 interaction was genotype dependent. Conclusions: Our data show that in thyroid, rs4644 is a trans-expression quantitative trait locus that can modify the transcriptional expression of downstream genes, through the modulation of TTF-1.

Drug-repositioning screening identified fludarabine and risedronic acid as potential therapeutic compounds for malignant pleural mesothelioma.

Objectives Malignant pleural mesothelioma (MPM) is an occupational disease mainly due to asbestos exposure. Effective therapies for MPM are lacking, making this tumour type a fatal disease. Materials and Methods In order to meet this need and in view of a future "drug repositioning" approach, here we screened five MPM (Mero-14, Mero-25, IST-Mes2, NCI-H28 and MSTO-211H) and one SV40-immortalized mesothelial cell line (MeT-5A) as a non-malignant model, with a library of 1170 FDA-approved drugs. Results Among several potential compounds, we found that fludarabine (F-araA) and, to a lesser extent, risedronic acid (RIS) were cytotoxic in MPM cells, in comparison to the non-malignant Met-5A cells. In particular, F-araA reduced the proliferation and the colony formation ability of the MPM malignant cells, in comparison to the non-malignant control cells, as demonstrated by proliferation and colony formation assays, in addition to measurement of the phospho-ERK/total-ERK ratio. We have shown that the response to F-araA was not dependent upon the expression of DCK and NT5E enzymes, nor upon their functional polymorphisms (rs11544786 and rs2295890, respectively). Conclusion This drug repositioning screening approach has identified that F-araA could be therapeutically active against MPM cells, in addition to other tumour types, by inhibiting STAT1 expression and nucleic acids synthesis. Further experiments are required to fully investigate this.

Variation rs2235503 C > A Within the Promoter of MSLN Affects Transcriptional Rate of Mesothelin and Plasmatic Levels of the Soluble Mesothelin-Related Peptide.

Soluble mesothelin-related peptide (SMRP) is a promising biomarker for malignant pleural mesothelioma (MPM), but several confounding factors can reduce SMRP-based test's accuracy. The identification of these confounders could improve the diagnostic performance of SMRP. In this study, we evaluated the sequence of 1,000 base pairs encompassing the minimal promoter region of the MSLN gene to identify expression quantitative trait loci (eQTL) that can affect SMRP. We assessed the association between four MSLN promoter variants and SMRP levels in a cohort of 72 MPM and 677 non-MPM subjects, and we carried out in vitro assays to investigate their functional role. Our results show that rs2235503 is an eQTL for MSLN associated with increased levels of SMRP in non-MPM subjects. Furthermore, we show that this polymorphic site affects the accuracy of SMRP, highlighting the importance of evaluating the individual's genetic background and giving novel insights to refine SMRP specificity as a diagnostic biomarker.

EIF4G1 and RAN as Possible Drivers for Malignant Pleural Mesothelioma.

For malignant pleural mesothelioma (MPM) novel therapeutic strategies are urgently needed. In a previous study, we identified 51 putative cancer genes over-expressed in MPM tissues and cell lines. Here, we deepened the study on nine of them (ASS1, EIF4G1, GALNT7, GLUT1, IGF2BP3 (IMP3), ITGA4, RAN, SOD1, and THBS2) to ascertain whether they are truly mesothelial cancer driver genes (CDGs) or genes overexpressed in an adaptive response to the tumoral progression ("passenger genes"). Through a fast siRNA-based screening, we evaluated the consequences of gene depletion on migration, proliferation, colony formation capabilities, and caspase activities of four MPM (Mero-14, Mero-25, IST-Mes2, and NCI-H28) and one SV40-immortalized mesothelial cell line (MeT-5A) as a non-malignant model. The depletion of EIF4G1 and RAN significantly reduced cell proliferation and colony formation and increased caspase activity. In particular, the findings for RAN resemble those observed for other types of cancer. Thus, we evaluated the in vitro effects of importazole (IPZ), a small molecule inhibitor of the interaction between RAN and importin-ß. We showed that IPZ could have effects similar to those observed following RAN gene silencing. We also found that primary cell lines from one out of three MPM patients were sensitive to IPZ. As EIF4G1 and RAN deserve further investigation with additional in vitro and in vivo studies, they emerged as promising CDGs, suggesting that their upregulation could play a role in mesothelial tumorigenesis and aggressiveness. Furthermore, present data propose the molecular pathways dependent on RAN as a putative pharmacological target for MPM patients in the view of a future personalized medicine.

Polymorphisms Within the RET Proto-Oncogene and Risk of Sporadic Medullary Thyroid Carcinoma.

Background: Sporadic medullary thyroid carcinoma (sMTC) is an uncommon neoplasia arising from the calcitonin-producing parafollicular cells of the thyroid. Previous studies evaluated whether single nucleotide polymorphisms (SNPs) within RET (a pivotal proto-oncogene for this disease) are associated with the risk for developing sMTC, but the results are inconclusive. Methods: In this work, we evaluated the association of RET-SNPs c.74-126G>T (rs2565206), p.Gly691Ser (rs1799939, G>A), p.Leu769 = (rs1800861, G>T), p.Ser836 = (rs1800862, C>T), and p.Ser904 = (rs1800863, C>G) (listed in the order of their chromosomal location) with sMTC. This is one of the largest case-control association studies carried out on sMTC, including 585 sMTC cases (negative for germline mutations within RET), 1529 patients affected by sporadic nonmedullary thyroid carcinoma (sNMTC), and 989 healthy controls, from central and southern Italy and collected in the period 2000-2017. Results: sNMTC patients showed similar genotype and allele frequencies compared with healthy controls. On the other hand, among sMTC patients, the T-allele of p.Leu769 = was less frequent (OR = 0.70 [CI 0.58-0.84], p = 1.9 × 10-4) and rare homozygotes TT showed an OR = 0.32 ([CI 0.17-0.60], p = 2.3 × 10-4). Moreover, a statistically significant excess of the haplotype 2 (characterized by the alleles T-G-G-C-C of the listed SNPs) was observed (p = 3.9 × 10-3). The SNPs were not associated with the expression of RET mRNA, that is, they did not exert an effect in cis as quantitative trait locus (cis-eQTL). However, a strong eQTL association was found for p.Leu769 = and the neighboring gene CSGALNACT2 (p = 9.3 × 10-50; effect-size = -0.65), whose function in cancer is unknown. Conclusions: This study shows that specific RET haplotypes, in particular haplotype 2 and the T-allele of p.Leu769 = , are associated with a reduced risk of sMTC in Italians.

Functional polymorphism within NUP210 encoding for nucleoporin GP210 is associated with the risk of endometriosis.

OBJECTIVE: To investigate whether nucleoporin 210 (GP210, encoded by NUP210 gene) is involved in endometriosis. DESIGN: Immunohistofluorescence analysis for assessing whether GP210 is expressed in endometrial tissues from patients and controls; genotyping and case-control study for assessing the association between rs354476 within NUP210 and risk of endometriosis; in vitro luciferase assay for assessing the functional activity of rs354476. SETTING: University. PATIENT(S): Histologically diagnosed cases (n = 175) of endometriosis: minimal or mild (stage I-II) in 48 cases (28%), moderate (stage III) in 69 cases (39%), and severe (stage IV) in 58 cases (33%). Controls (n = 557) were female blood donors collected at Meyer Hospital of Florence. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): GP210 tissue expression; genotype distribution and risk of endometriosis; in vitro gene expression measurements. RESULT(S): GP210 had positive nuclear immunohistofluorescence staining in endometrial glandular epithelium. Carriers of the variant allele were associated with increased risks: C/T, odds ratio (OR) 1.83, 95% confidence interval (CI) 1.04-3.21; T/T, OR 2.55, 95% CI 1.36-4.80. In vitro, luciferase assay showed that rs354476 is a bona fide target for hsa-miR-125b-5p. CONCLUSION(S): Nucleoporin GP210 is involved in endometriosis. Rs354476 polymorphism affects the regulation of NUP210 gene expression by altering the binding with hsa-miR-125b-5p, a microRNA already known as playing an important role for endometriosis. This provides the rationale for the observed increased risk of endometriosis in carriers of the variant allele.

Correction: Inherited variants in genes somatically mutated in thyroid cancer.

[This corrects the article DOI: 10.1371/journal.pone.0174995.].

The polymorphism rs2480258 within CYP2E1 is associated with different rates of acrylamide metabolism in vivo in humans.

In a recent study, we demonstrated that the variant allele of rs2480258 within intron VIII of CYP2E1 is associated with reduced levels of mRNA, protein, and enzyme activity. CYP2E1 is the most important enzyme in the metabolism of acrylamide (AA) by operating its oxidation into glycidamide (GA). AA occurs in food, is neurotoxic and classified as a probable human carcinogen. The goal of the present study was to further assess the role of rs2480258 by measuring the rate of AA > GA biotransformation in vivo. In blood samples from a cohort of 120 volunteers, the internal doses of AA and GA were assessed by AA and GA adducts to hemoglobin (Hb) measured by mass spectrometry. The rate of biotransformation was assessed by calculating the GA-Hb/AA-Hb ratio. To maximize the statistical power, 60 TT was compared to 60 CC-homozygotes and the results showed that TT homozygotes had a statistically significant reduced rate of biotransformation. Present results reinforced the notion that T-allele of rs2480258 is a marker of low functional activity of CYP2E1. Moreover, we studied the role of polymorphisms (SNPs) within glutathione-S-transferases (GSTs) enzymes and epoxide hydrolase (EPHX), verifying previous findings that SNPs within GSTs and EPHX influence the metabolism rate.

Deregulation of miRNAs in malignant pleural mesothelioma is associated with prognosis and suggests an alteration of cell metabolism.

Malignant pleural mesothelioma (MPM) is an aggressive human cancer and miRNAs can play a key role for this disease. In order to broaden the knowledge in this field, the miRNA expression was investigated in a large series of MPM to discover new pathways helpful in diagnosis, prognosis and therapy. We employed nanoString nCounter system for miRNA profiling on 105 MPM samples and 10 healthy pleura. The analysis was followed by the validation of the most significantly deregulated miRNAs by RT-qPCR in an independent sample set. We identified 63 miRNAs deregulated in a statistically significant way. MiR-185, miR-197, and miR-299 were confirmed differentially expressed, after validation study. In addition, the results of the microarray analysis corroborated previous findings concerning miR-15b-5p, miR-126-3p, and miR-145-5p. Kaplan-Meier curves were used to explore the association between miRNA expression and overall survival (OS) and identified a 2-miRNA prognostic signature (Let-7c-5p and miR-151a-5p) related to hypoxia and energy metabolism respectively. In silico analyses with DIANA-microT-CDS highlighted 5 putative targets in common between two miRNAs. With the present work we showed that the pattern of miRNAs expression is highly deregulated in MPM and that a 2-miRNA signature can be a new useful tool for prognosis in MPM.

Inhibition of the platelet-derived growth factor receptor beta (PDGFRB) using gene silencing, crenolanib besylate, or imatinib mesylate hampers the malignant phenotype of mesothelioma cell lines.

Malignant pleural mesothelioma (MPM) is a cancer of the pleural cavity resistant to chemotherapy. The identification of novel therapeutic targets is needed to improve its poor prognosis. Following a review of literature and a screening of specimens we found that platelet-derived growth factor receptor beta (PDGFRB) is over-expressed, but not somatically mutated, in MPM tissues. We aimed to ascertain whether PDGFRB is a MPM-cancer driver gene. The approaches employed included the use of gene silencing and the administration of small molecules, such as crenolanib and imatinib (PDGFR inhibitors) on MPM cell lines (IstMes2, Mero-14, Mero-25). Met5A cells were used as non-malignant mesothelial cell line. PDGFRB-silencing caused a decrease in the proliferation rate, and a reduced colony formation capacity, as well as an increase of the share of cells in sub-G1 and in G2 phase, and increased apoptotic rate of MPM cell lines. Loss of migration ability was also observed. Similar, or even further enhanced, results were obtained with crenolanib. Imatinib showed the least effective activity on the phenotype. In conclusion, our study highlights PDGFRB as target with a clear role in MPM tumorigenesis and provided a rationale to explore further the efficacy of crenolanib in MPM patients, with promising results.

Inherited variants in genes somatically mutated in thyroid cancer.

BACKGROUND: Tumour suppressor genes when mutated in the germline cause various cancers, but they can also be somatically mutated in sporadic tumours. We hypothesized that there may also be cancer-related germline variants in the genes commonly mutated in sporadic well-differentiated thyroid cancer (WDTC). METHODS: We performed a two-stage case-control association study with a total of 2214 cases and 2108 healthy controls from an Italian population. By genotyping 34 single nucleotide polymorphisms (SNPs), we covered a total of 59 missense SNPs and SNPs located in the 5' and 3' untranslated regions (UTRs) of 10 different genes. RESULTS: The Italian1 series showed a suggestive association for 8 SNPs, from which three were replicated in the Italian2 series. The meta-analysis revealed a study-wide significant association for rs459552 (OR: 0.84, 95%CI: 0.75-0.94) and rs1800900 (OR: 1.15, 95%CI: 1.05-1.27), located in the APC and GNAS genes, respectively. The APC rs459552 is a missense SNP, located in a conserved amino acid position, but without any functional consequences. The GNAS rs1800900 is located at a conserved 5'UTR and according to the experimental ENCODE data it may affect promoter and histone marks in different cell types. CONCLUSIONS: The results of this study yield new insights on WDTC, showing that inherited variants in the APC and GNAS genes can play a role in the etiology of thyroid cancer. Further studies are necessary to better understand the role of the identified SNPs in the development of WDTC and to functionally validate our in silico predictions.

Mesothelin promoter variants are associated with increased soluble mesothelin-related peptide levels in asbestos-exposed individuals.

BACKGROUND: Soluble mesothelin-related peptide (SMRP) is a promising diagnostic biomarker for malignant pleural mesothelioma (MPM), but various confounders hinder its usefulness in surveillance programmes. We previously showed that a single nucleotide polymorphism (SNP) within the 3'untranslated region (3'UTR) of the mesothelin (MSLN) gene could affect the levels of SMRP. OBJECTIVES: To focus on SNPs located within MSLN promoter as possible critical genetic variables in determining SMRP levels. METHODS: The association between SMRP and SNPs was tested in 689 non-MPM subjects and 70 patients with MPM. Reporter plasmids carrying the four most common haplotypes were compared in a dual luciferase assay, and in silico analyses were performed to investigate the putative biological role of the SNPs. RESULTS: We found a strong association between serum SMRP and variant alleles of rs3764247, rs3764246 (in strong linkage disequilibrium with rs2235504) and rs2235503 in non-MPM subjects. Inclusion of the genotype information led to an increase in SMRP specificity from 79.9% to 85.5%. Although not statistically significant, the group with MPM showed the same trend of association. According to the in vitro luciferase study, rs3764247 itself had a functional role. In silico approaches showed that the binding sites for transcription factors such as Staf and ZNF143 could be affected by this SNP. The other SNPs were shown to interact with each other in a more complex way. CONCLUSIONS: These data support the suggestion that SMRP performance is affected by individual (ie, genetic) variables and that MSLN expression is influenced by SNPs located within the promoter regulatory region.

Bonafide Targets of Deregulated microRNAs in Non-Small Cell Lung Cancer as Tool to Identify Novel Therapeutic Targets: A Review.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is an aggressive neoplasm with a poor survival and novel therapies are urgently needed. The study of deregulated micro- RNAs (dereg-miRs) could constitute a strategy helping to detect specific genes playing a relevant role in the disease. Thus, the oncoproteins encoded by these genes could be exploited as novel therapeutic targets to be inhibited by small molecules, aptamers, or monoclonal antibodies. METHODS: The present review is focused on candidate genes having convincing biological evidences to be both bona fide targets for dereg-miRs and playing a role in NSCLC progression. These genes were evaluated according to the molecular pathway they belong. Moreover, in the attempt to provide an even broader list of candidate therapeutic targets for NSCLC, the full list of genes was analyzed using the online tool Interactome DB. RESULTS: Among the identified targets, some of them belong to p53 or MAP kinase signaling pathways, and others include caspases, MCL1, and BCL2L2 (playing a role in apoptosis), ZEB1, ZEB2, and USP25 (epithelial-to-mesenchymal transition), EZH2, SOX9, and HOXA5 (differentiation), Paxillin, LIMK1 and MTDH (cytoskeleton remodeling), and HDGF (angiogenesis). In addition, other targets, such as TIMP-2, PIM-1, and components of the IGF-signaling pathways were suggested following the interactome analysis. CONCLUSION: Studies on dereg-miRs helped to identify a set of genes whose encoded proteins could constitute candidates for future therapeutic approaches.

Polymorphisms within base and nucleotide excision repair pathways and risk of differentiated thyroid carcinoma.

The thyrocytes are exposed to high levels of oxidative stress which could induce DNA damages. Base excision repair (BER) is one of the principal mechanisms of defense against oxidative DNA damage, however recent evidences suggest that also nucleotide excision repair (NER) could be involved. The aim of present work was to identify novel differentiated thyroid cancer (DTC) risk variants in BER and NER genes. For this purpose, the most strongly associated SNPs within NER and BER genes found in our previous GWAS on DTC were selected and replicated in an independent series of samples for a new case-control study. Although a positive signal was detected at the nominal level of 0.05 for rs7689099 (encoding for an aminoacid change proline to arginine at codon 117 within NEIL3), none of the considered SNPs (i.e. rs7990340 and rs690860 within RFC3, rs3744767 and rs1131636 within RPA1, rs16962916 and rs3136166 in ERCC4, and rs17739370 and rs7689099 in NEIL3) was associated with the risk of DTC when the correction of multiple testing was applied. In conclusion, a role of NER and BER pathways was evoked in the susceptibility to DTC. However, this seemed to be limited to few polymorphic genes and the overall effect size appeared weak.

Runs of homozygosity and inbreeding in thyroid cancer.

BACKGROUND: Genome-wide association studies (GWASs) have identified several single-nucleotide polymorphisms (SNPs) influencing the risk of thyroid cancer (TC). Most cancer predisposition genes identified through GWASs function in a co-dominant manner, and studies have not found evidence for recessively functioning disease loci in TC. Our study examines whether homozygosity is associated with an increased risk of TC and searches for novel recessively acting disease loci. METHODS: Data from a previously conducted GWAS were used for the estimation of the proportion of phenotypic variance explained by all common SNPs, the detection of runs of homozygosity (ROH) and the determination of inbreeding to unravel their influence on TC. RESULTS: Inbreeding coefficients were significantly higher among cases than controls. Association on a SNP-by-SNP basis was controlled by using the false discovery rate at a level of q* < 0.05, with 34 SNPs representing true differences in homozygosity between cases and controls. The average size, the number and total length of ROHs per person were significantly higher in cases than in controls. A total of 16 recurrent ROHs of rather short length were identified although their association with TC risk was not significant at a genome-wide level. Several recurrent ROHs harbor genes associated with risk of TC. All of the ROHs showed significant evidence for natural selection (iHS, Fst, Fay and Wu's H). CONCLUSIONS: Our results support the existence of recessive alleles in TC susceptibility. Although regions of homozygosity were rather small, it might be possible that variants within these ROHs affect TC risk and may function in a recessive manner.

A Comprehensive Meta-analysis of Case-Control Association Studies to Evaluate Polymorphisms Associated with the Risk of Differentiated Thyroid Carcinoma.

BACKGROUND: Linkage analyses and association studies suggested that inherited genetic variations play a role in the development of differentiated thyroid carcinoma (DTC). METHODS: We combined the results from a genome-wide association study (GWAS) performed by our group and from published studies on DTC. With a first approach, we evaluated whether a SNP published as associated with the risk of DTC could replicate in our GWAS (using FDR as adjustment for multiple comparisons). With the second approach, meta-analyses were performed between literature and GWAS when both sources suggested an association, increasing the statistical power of the analysis. RESULTS: rs1799814 (CYP1A1), rs1121980 (FTO), and 3 SNPs within 9q22 (rs965513, rs7048394, and rs894673) replicated the associations described in the literature. In addition, the meta-analyses between literature and GWAS revealed 10 more SNPs within 9q22, six within FTO, two within SOD1, and single variations within HUS1, WDR3, UGT2B7, ALOX12, TICAM1, ATG16L1, HDAC4, PIK3CA, SULF1, IL11RA, VEGFA, and 1p31.3, 2q35, 8p12, and 14q13. CONCLUSION: This analysis confirmed several published risk loci that could be involved in DTC predisposition. IMPACT: These findings provide evidence for the role of germline variants in DTC etiology and are consistent with a polygenic model of the disease. Cancer Epidemiol Biomarkers Prev; 25(4); 700-13. ©2016 AACR.

Association between CYP2E1 polymorphisms and risk of differentiated thyroid carcinoma.

Differentiated thyroid carcinoma (DTC) results from complex interactions between genetic and environmental factors. Known etiological factors include exposure to ionizing radiations, previous thyroid diseases, and hormone factors. It has been speculated that dietary acrylamide (AA) formed in diverse foods following the Maillard's reaction could be a contributing factor for DTC in humans. Upon absorption, AA is biotransformed mainly by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA). Considering that polymorphisms within CYP2E1 were found associated with endogenous levels of AA-Valine and GA-Valine hemoglobin adducts in humans, we raised the hypothesis that specific CYP2E1 genotypes could be associated with the risk of DTC. Analysis of four haplotype tagging SNPs (ht-SNPs) within the locus in a discovery case-control study (N = 350/350) indicated an association between rs2480258 and DTC risk. This ht-SNP resides within a linkage disequilibrium block spanning intron VIII and the 3'-untranslated region. Extended analysis in a large replication set (2429 controls and 767 cases) confirmed the association, with odds ratios for GA and AA genotypes of 1.24 (95 % confidence interval (CI) 1.03-1.48) and 1.56 (95 % CI, 1.06-2.30), respectively. Functionally, the minor allele was associated with low levels of CYP2E1 mRNA and protein expression as well as lower enzymatic activity in a series of 149 human liver samples. Our data support the hypothesis that inter-individual differences in CYP2E1 activity could modulate the risk of developing DTC suggesting that the exposure to specific xenobiotics, such as AA, could play a role in this process.

Novel genetic variants in differentiated thyroid cancer and assessment of the cumulative risk.

A genome-wide association study (GWAS) performed on a high-incidence Italian population followed by replications on low-incidence cohorts suggested a strong association of differentiated thyroid cancer (DTC) with single nucleotide polymorphisms (SNPs) at 9q22.33, 2q35, 20q11.22-q12 and 14q24.3. Moreover, six additional susceptibility loci were associated with the disease only among Italians. The present study had two aims, first to identify loci involved in DTC risk and then to assess the cumulative effect of the SNPs identified so far in the Italian population. The combined analysis of the previous GWAS and the present Italian study provided evidence of association with rs7935113 (GALNTL4, OR = 1.36, 95%CI 1.20-1.53, p-value = 7.41 × 10(-7)) and rs1203952 (FOXA2, OR = 1.29, 95%CI 1.16-1.44, p-value = 4.42 × 10(-6)). Experimental ENCODE and eQTL data suggested that both SNPs may influence the closest genes expression through a differential recruitment of transcription factors. The assessment of the cumulative risk of eleven SNPs showed that DTC risk increases with an increasing number of risk alleles (p-trend = 3.13 × 10(-47)). Nonetheless, only a small fraction (about 4% on the disease liability scale) of DTC is explained by these SNPs. These data are consistent with a polygenic model of DTC predisposition and highlight the importance of association studies in the discovery of the disease hereditability.