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Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. The molecular characteristics of histologically normal appearing tissue adjacent to the tumor (NAT) from PTC patients are not well characterized. The aim of this study was to characterize the global gene expression profile of NAT and compare it with those of normal and tumor thyroid tissues. We performed total RNA sequencing with fresh frozen thyroid tissues from a cohort of three categories of samples including NAT, normal thyroid (N), and PTC tumor (T). Transcriptome analysis shows that NAT presents a unique gene expression profile, which was not associated with sex or the presence of lymphocytic thyroiditis. Among the differentially expressed genes (DEGs) of NAT vs N, 256 coding genes and 5 noncoding genes have been reported as cancer genes involved in cell proliferation, apoptosis, and/or tumorigenesis. Bioinformatics analysis with Ingenuity Pathway Analysis software revealed that “Cancer, Organismal Injury and Abnormalities, Cellular Response to Therapeutics, and Cellular Movement” were major dysregulated pathways in the NAT tissues. This study provides improved insight into the complexity of gene expression changes in the thyroid glands of patients with PTC.

BackgroundPapillary thyroid carcinoma (PTC) demonstrates high heritability and a low somatic mutation burden relative to other cancers. Therefore, the genetic risk predisposing to PTC is likely due to a combination of low penetrance variants. A recent genome-wide association study revealed the association of PTC with a missense variant, rs6793295, at 3q26 in a gene called Leucine Repeat Rich Containing 34 (LRRC34).MethodsWe report the mechanisms of PTC risk at 3q26 using a combination of overexpression, mass spectroscopy, knockdown, transcriptome profiling, migration assays and genetic analysis.ResultsWe observed differential binding of wild-type and missense LRRC34 to RANBP1. Overexpression of missense LRRC34 reduced RanGTP levels and increased apoptosis. We also identified a second linkage disequilibrium (LD) block upstream of LRRC34 containing regulatory variants with allele-specific expression. Transcriptome profiling of LRRC34 knockdown cells showed changes in genes involved with cellular movement. LRRC34 knockdown reduced the migration of thyroid cancer cell lines. Lastly, we assessed the relative contribution of PTC risk from each locus using haplotype analysis.ConclusionsOur study demonstrates two separate mechanisms, one in G protein signalling and the other in transcriptional control, dictating PTC risk at 3q26 using both biochemical and genetic techniques.

Background: Our genome sequencing analysis revealed a frameshift mutation in the shelterin gene TINF2 in a large family with individuals affected with papillary thyroid carcinoma (PTC) and melanoma. Here, we further characterized the mutation and screened for coding variants in the 6 shelterin genes in 24 families. Methods: Sanger sequencing was performed to screen for the TINF2 mutation in the key family. Quantitative reverse transcription-polymerase chain reaction (PCR) was used for TINF2 gene expression analysis. Exogenous expression and co-immunoprecipitation techniques were used for assessing TINF2 binding to TERF1. Relative telomere length (RTL) was quantified in DNAs from lymphocytes by using quantitative real-time PCR. Whole exome sequencing (WES) was performed in seven families with individuals affected with PTC and other cancer types. Screening for DNA variants in shelterin genes was performed by using whole genome sequencing data from 17 families and WES data from 7 further families. Results: The TINF2 mutation (TINF2 p.Trp198fs) showed complete co-segregation with PTC and melanoma in the key family. The mutation is not reported in databases and not identified in 23 other families we screened. The expression of TINF2 was borderline reduced in individuals with the mutation. The truncated TINF2 protein showed abolished binding to TERF1. The RTL in the individuals with the mutation was significantly longer when compared with those without the mutation from the same family as well as compared with 62 healthy controls. Among the 24 families, we identified 3 missense and 1 synonymous variant(s) in 2 shelterin genes (TINF2 and ACD). Conclusions: The rare frameshift mutation in the TINF2 gene and the associated longer telomere length suggest that dysregulated telomeres could be a mechanism predisposing to PTC and melanoma. DNA coding variants in shelterin genes are rare. Further studies are required to evaluate the roles of variants in shelterin genes in thyroid cancer and melanoma.

Background We studied a large family with 22 individuals affected with autosomal dominant hereditary spherocytosis (HS). Methods Genome‐wide linkage, whole‐genome sequencing (WGS), Sanger sequencing, RT‐PCR, and ToPO TA cloning analyses were performed. Results We revealed a heterozygous G>A transition in the 14q23 locus, at position +1 of the intron 8 donor splice site of the spectrin beta, erythrocytic (SPTB) gene. This splice variant (SPTB c.1064+1G>A) was confirmed by Sanger sequencing and showed complete co‐segregation with HS in the family. Further RT‐PCR reactions and sequencing analysis indicated that the variant leads to the exclusion of exon 8 and subsequent frameshift in exon 9 and a premature stop codon in SPTB. Translation of the altered allele would lead to a truncation with a loss of all spectrin repeat domains in SPTB protein. Conclusion This variant is novel and has not been found in any databases. We propose that this splice variant explains the spherocytosis phenotype observed in this large family., We studied a large family with 22 individuals affected with autosomal dominant hereditary spherocytosis (HS). Genome‐wide linkage and whole‐genome sequencing analyses revealed a heterozygous G>A transition in the 14q23 locus, at position +1 of the intron 8 donor splice site of the spectrin beta, erythrocytic (SPTB) gene.

Incidence of differentiated thyroid carcinoma (DTC) varies considerably between ethnic groups, with particularly high incidence rates in Pacific Islanders. Here, we conducted a genome-wide association study (GWAS) involving 1,554 cases/1,973 controls of European ancestry and 301 cases/348 controls of Oceanian ancestry from the EPITHYR consortium. Our results confirmed the association with the known DTC susceptibility loci at 2q35, 8p12, 9q22.33 and 14q13.3 in the European ancestry population and suggested two novel signals at 1p31.3 (rs334729) and 16q23.2 (rs16950982), which were associated with TSH levels in previous GWAS. We additionally replicated an association with 5p15.33 reported previously in Chinese and European populations. Except at 1p31.3, all associations were in the same direction in the population of Oceanian ancestry. The frequency of risk alleles at 2q35, 5p15.33 and 16q23.2 were significantly higher in Oceanians than in Europeans and may explain part of the highest DTC incidence observed in Oceanians.Competing Interest StatementThe authors have declared no competing interest.Funding StatementINCA (grant number 9533) and ARC (grant number PGA120150202302), Ligue Nationale Contre le Cancer, Agence Nationale pour la Recherche (ANR), the Direction Generale de la Sante, the Agence Francaise de Securite Sanitaire de l alimentation, de lenvironnement et du travail (ANSES), CHILDTHYR EEC program, and the Fondation de France. JG and CX were the recipient of a PhD fellowship from Region Ile-de-France.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:All participants provided informed consent and each study was approved by their governing ethics committee.All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesThe data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. (Less)

Papillary thyroid carcinoma (PTC) is the most common histotype of thyroid carcinoma. The heritability of PTC is high compared to other cancers, but its underlying causes are unknown. A recent genome-wide association study revealed the association of a variant at the 5q22 locus, rs73227498, with PTC predisposition. We report that rs17134155, a variant in high linkage disequilibrium with rs73227498, is located in an enhancer region downstream of coding transcripts of EPB41L4A. Rs17134155 showed significant enhancer activity in luciferase assays, and haplotypes containing the protective allele of this variant conferred a significantly lower risk of PTC. While the index SNP, rs73227498, acted as a significant cis-eQTL for expression of EPB41L4A, rs17134155 was a significant cis-sQTL for the alternative splicing of a non-coding transcript of EPB41L4A, called EPB41L4A-203. We also performed knockdown of EPB41L4A followed by microarray analysis. Some of the top differentially-expressed genes were represented among regulators of the WNT/β-catenin signaling pathway. Our results indicate that an enhancer region at 5q22 regulates the expression and splicing of EPB41L4A transcripts. We also provide evidence that EPB41L4A expression is involved in regulating growth and differentiation pathways, suggesting that decreased expression of EPB41L4A is a mechanism in the predisposition to PTC.

To identify and characterize the functional variants, regulatory gene networks, and potential binding targets of SMAD3 in the 15q22 thyroid cancer risk locus. We performed linkage disequilibrium (LD) and haplotype analyses to fine map the 15q22 locus. Luciferase reporter assays were applied to evaluate the regulatory effects of the candidate variants. Knockdown by small interfering RNA, microarray analysis, chromatin immunoprecipitation (ChIP) and quantitative real-time polymerase chain reaction assays were performed to reveal the regulatory gene network and identify its binding targets. We report a 25.6-kb haplotype within SMAD3 containing numerous single-nucleotide polymorphisms (SNPs) in high LD. SNPs rs17293632 and rs4562997 were identified as functional variants of SMAD3 by luciferase assays within the LD region. These variants regulate SMAD3 transcription in an allele-specific manner through enhancer elements in introns of SMAD3. Knockdown of SMAD3 in thyroid cancer cell lines revealed its regulatory gene network including two upregulated genes, SPRY4 and SPRY4-IT1. Sequence analysis and ChIP assays validated the actual binding of SMAD3 protein to multiple SMAD binding element sites in the region upstream of SPRY4. Our data provide a functional annotation of the 15q22 thyroid cancer risk locus.

Background: Familial non-medullary thyroid cancer (NMTC) accounts for a relatively small proportion of thyroid cancer cases, but it displays strong genetic predisposition. So far, only a few NMTC susceptible genes and low-penetrance variants contributing to NMTC have been described. This study aimed to identify rare germline variants that may predispose individuals to NMTC by sequencing a cohort of 17 NMTC families. Methods: Whole-genome sequencing and genome-wide linkage analysis were performed in 17 NMTC families. MendelScan and BasePlayer were applied to screen germline variants followed by customized filtering. The remaining candidate variants were subsequently validated by Sanger sequencing. A panel of 277 known cancer predisposition genes was also screened in these families. Results: A total of 41 rare coding candidate variants in 40 genes identified by whole-genome sequencing are reported, including 24 missense, five frameshift, five splice change, and seven nonsense variants. Sanger sequencing confirmed all 41 rare variants and proved their co-segregation with NMTC in the extended pedigrees. In silico functional analysis of the candidate genes using Ingenuity Pathway Analysis showed that cancer was the top category of "Diseases and Disorders." Additionally, a targeted search displayed six variants in known cancer predisposition genes, including one frameshift variant and five missense variants. Conclusions: The data identify rare germline variants that may play important roles in NMTC predisposition. It is proposed that in future research including functional characterization, these variants and genes be considered primary candidates for thyroid cancer predisposition.

The [A] allele of SNP rs965513 in 9q22 has been consistently shown to be highly associated with increased papillary thyroid cancer (PTC) risk with an odds ratio of ∼1.8 as determined by genome-wide association studies, yet the molecular mechanisms remain poorly understood. Previously, we noted that the expression of two genes in the region, forkhead box E1 (FOXE1) and PTC susceptibility candidate 2 (PTCSC2), is regulated by rs965513 in unaffected thyroid tissue, but the underlying mechanisms were not elucidated. Here, we fine-mapped the 9q22 region in PTC and controls and detected an ∼33-kb linkage disequilibrium block (containing the lead SNP rs965513) that significantly associates with PTC risk. Chromatin characteristics and regulatory element signatures in this block disclosed at least three regulatory elements functioning as enhancers. These enhancers harbor at least four SNPs (rs7864322, rs12352658, rs7847449, and rs10759944) that serve as functional variants. The variant genotypes are associated with differential enhancer activities and/or transcription factor binding activities. Using the chromosome conformation capture methodology, long-range looping interactions of these elements with the promoter region shared by FOXE1 and PTCSC2 in a human papillary thyroid carcinoma cell line (KTC-1) and unaffected thyroid tissue were found. Our results suggest that multiple variants coinherited with the lead SNP and located in long-range enhancers are involved in the transcriptional regulation of FOXE1 and PTCSC2 expression. These results explain the mechanism by which the risk allele of rs965513 predisposes to thyroid cancer.

The great majority of thyroid cancers are of the non-medullary type. Here we report findings from a genome-wide association study of non-medullary thyroid cancer, including in total 3,001 patients and 287,550 controls from five study groups of European descent. Our results yield five novel loci (all with Pcombined, We thank the patients whose contribution made this work possible. The study was supported in part by grants from the National Institutes of Health: CA16058 (to the Ohio State University Comprehensive Cancer Center) and CA168505 (to M.D.R. and A.d.l.C.). This work was supported in part by The University of Texas MD Anderson Cancer Center start-up funds (to E.M.S.); an American Thyroid Association research grant (to E.M.S.), National Institutes of Health (NIH) grant U01 DE019765-01 (to E.M.S.); and Cancer Center Support Grant CA016672 to the University of Texas MD Anderson Cancer Center. This study makes use of data generated by the Genome of the Netherlands Project (GoNL). A full list of the investigators is available from http://www.nlgenome.nl. Funding for the GoNL project was provided by the Netherlands Organization for Scientific Research under award number 184021007, dated 9 July 2009 and made available as a Rainbow Project of the Biobanking and Biomolecular Research Infrastructure Netherlands (BBMRI-NL). The sequencing was carried out in collaboration with the Beijing Institute for Genomics (BGI). This study also makes use of data generated by the GTEx Consortium and we are grateful for early stage sharing of RNA-seq and genotypic data. The following acknowledgement refers to the GTEx data: The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health. Additional funds were provided by the NCI, NHGRI, NHLBI, NIDA, NIMH and NINDS. Donors were enrolled at Biospecimen Source Sites funded by NCI\SAIC-Frederick, Inc. (SAIC-F) subcontracts to the National Disease Research Interchange (10XS170), Roswell Park Cancer Institute (10XS171) and Science Care, Inc. (X10S172). The Laboratory, Data Analysis and Coordinating Center (LDACC) was funded through a contract (HHSN268201000029C) to The Broad Institute, Inc. Biorepository operations were funded through an SAIC-F subcontract to Van Andel Institute (10ST1035). Additional data repository and project management were provided by SAIC-F (HHSN261200800001E). The Brain Bank was supported by supplements to University of Miami grants DA006227 and DA033684 and to contract N01MH000028. Statistical Methods development grants were made to the University of Geneva (MH090941 and MH101814), the University of Chicago (MH090951, MH090937, MH101820 and MH101825), the University of North Carolina—Chapel Hill (MH090936 and MH101819), Harvard University (MH090948), Stanford University (MH101782), Washington University St Louis (MH101810) and the University of Pennsylvania (MH101822).

// Jerneja Tomsic 1, 7 , Rebecca Fultz 1 , Sandya Liyanarachchi 1 , Luke K. Genutis 1 , Yanqiang Wang 1 , Wei Li 1 , Stefano Volinia 2 , Krystian Jazdzewski 3, 4 , Huiling He 1 , Paul E Wakely Jr. 5 , Leigha Senter 6 , Albert de la Chapelle 1 1 Department of Cancer Biology and Genetics, The Ohio State University Wexner Medical Center and Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 2 Deptartment of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 3 Genomic Medicine, Medical University of Warsaw, Warsaw, Poland 4 Laboratory of Human Cancer Genetics, Centre of New Technologies, CENT, University of Warsaw, Warsaw, Poland 5 Department of Pathology, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, USA 6 Department of Internal Medicine, The Ohio State University Wexner Medical Center and Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA 7 Division of Biomarkers Early Detection Prevention, City of Hope, Duarte, CA, USA Correspondence to: Albert de la Chapelle, email: albert.delachapelle@osumc.edu Keywords: genetics, predisposition, miRNA, thyroid, variants Received: July 15, 2016 Accepted: December 16, 2016 Published: December 23, 2016 ABSTRACT Papillary Thyroid Carcinoma (PTC) displays one of the highest familiality scores of all cancers as measured by case-control studies, yet only a handful of genes have been implicated until now. Variants in microRNAs have been associated with the risk of several cancers including PTC but the magnitude of this involvement is unclear. This study was designed to test to what extent genomic variants in microRNAs contribute to PTC risk. We used SOLiD technology to sequence 321 genomic regions encoding 427 miRNAs in one affected individual from each of 80 PTC families. After excluding variants with frequency ≥ 1% in 1000 Genomes Phase 1 ( n = 1092) we detected 1978 variants. After further functional filtering steps 25 variants in pre-miRs remained. Co-segregation was observed for six out of 16 tested miRNA variants with PTC in the families, namely let-7e, miR-181b, miR-135a, miR-15b, miR-320, and miR-484. Expression of miR-135a and miR-181b was tested in normal thyroid and tumor tissue from patients that carry the variants and a decrease in expression was observed. In vitro assays were applied to measure the effect of the variants on microRNAs’ maturation. Four out of six variants were tested. Only the let-7e and miR-181b variants showed an effect on processing leading to lower levels of mature miRNA. These two variants were not detected in 1170 sporadic PTC cases nor in 1404 controls. Taken together, our data show that high penetrance germline sequence variants of miRNAs potentially predispose to a fraction of all PTC but are not common.

Papillary thyroid carcinoma (PTC) displays higher heritability than most other cancers. To search for genes predisposing to PTC, we performed a genome-wide linkage analysis in a large family with PTC and melanoma. Among several peaks the highest was at 8q24, with a maximum nonparametric linkage (NPL) score of 7.03. Linkage analysis was then broadened to comprise 25 additional PTC families that produced a maximum NPL score of 3.2, P = 0.007 at the 8q24 locus. Fine mapping with microsatellite markers was compatible with linkage to the 8q24 locus in 10 of the 26 families. In the large family, a ∼320 Kb haplotype was shared by individuals with PTC, melanoma, or benign thyroid disease, but not by unaffected individuals. A 12 Kb haplotype of 8 SNP markers within the larger haplotype was shared by 9 of the 10 families in which the 8q24 locus was compatible with linkage. The shared haplotype is located within 2 known overlapping protein-coding genes, thyroglobulin (TG) and Src-like adaptor (SLA). Resequencing of the coding and control regions of TG and SLA did not disclose putative mutations in PTC patients. Embedded in the TG-SLA region are three likely noncoding RNA genes, one of which (AK023948) harbors the 8-SNP haplotype. Resequencing of AK023948 and one of the other RNA genes did not reveal candidate mutations. Gene expression analysis indicated that AK023948 is significantly down-regulated in most PTC tumors. The putative noncoding RNA gene AK023948 is a candidate suseptibility gene for PTC. [Cancer Res 2009;69(2):625–31]

Papillary thyroid carcinoma (PTC) displays strong but so far largely uncharacterized heritability. Here we studied genetic predisposition in a family with six affected individuals. We genotyped all available family members and conducted whole exome sequencing of blood DNA from two affected individuals. Haplotype analysis and other genetic criteria narrowed our list of candidates to a germline variant in the serine/arginine repetitive matrix 2 gene (SRRM2). This heterozygous variant, c.1037C > T (Ser346Phe or S346F; rs149019598) cosegregated with PTC in the family. It was not found in 138 other PTC families. It was found in 7/1,170 sporadic PTC cases and in 0/1,404 controls (p = 0.004). The encoded protein SRRM2 (also called SRm300) is part of the RNA splicing machinery. To evaluate the possibility that the S346F missense mutation affects alternative splicing, we compared RNA-Seq data in leukocytes from three mutation carriers and three controls. Significant differences in alternative splicing were identified for 1,642 exons, of which a subset of 7 exons was verified experimentally. The results confirmed a higher ratio of inclusion of exons in mutation carriers. These data suggest that the S346F mutation in SRRM2 predisposes to PTC by affecting alternative splicing of unidentified downstream target genes.

Release of calcium from the endoplasmic reticulum (ER) signals an increase in transcription of both the early response gene, c-fos, and the late response gene, grp78. We have used thapsigargin (TG), an ER calcium-ATPase pump inhibitor that induces calcium release from the ER, to investigate the possible involvement of c-Fos, a component of the AP-1 transcription factor, in grp78 induction. Two cell lines with markedly different responses to TG treatment were employed: the WEHI7.2 mouse lymphoma line in which TG fails to induce grp78, and the MDA-MB-468 mammary epithelial line in which TG induces grp78. In WEHI7.2 cells, TG-induced calcium release triggers a rapid increase in c-fos mRNA, but the level of c-Fos protein decreases due to degradation by the multicatalytic proteasome. C-FosdeltaC, a proteasome resistant c-Fos mutant with AP-1 activity similar to that of wild type c-Fos, restores grp78 induction in WEHI7.2 cells, detected by both Northern hybridization and a grp78 promoter-luciferase reporter assay. In MDA-MB-468 cells, TG-mediated calcium release induces a sustained elevation of c-Fos protein that precedes grp78 induction. A region of the grp78 promoter containing both ERSE and CORE regions, but missing TRE and CRE regions, is sufficient to mediate induction of reporter luciferase activity. Induction of this reporter was blocked by A-Fos, a dominant negative inhibitor of c-Fos. Also, the induction of grp78-luciferase reporter activity was inhibited by c-fos antisense mRNA. In summary, the findings indicate that c-Fos is involved in signaling grp78 induction following TG treatment, and that grp78 induction is inhibited by proteasome-mediated c-Fos degradation.

Autoimmune thyroid diseases (AITD) are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune hypothyroidism (Hashimoto's thyroiditis), as well as autoimmune hyperthyroidism (Graves' disease). As the possible causative genes of TPOAbs and AITD remain largely unknown, we performed GWAS meta-analyses in 18,297 individuals for TPOAb-positivity (1769 TPOAb-positives and 16,528 TPOAb-negatives) and in 12,353 individuals for TPOAb serum levels, with replication in 8,990 individuals. Significant associations (P, Author Summary Individuals with thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune thyroid diseases (AITD), which are common in the general population and associated with increased cardiovascular, metabolic and psychiatric morbidity and mortality. As the causative genes of TPOAbs and AITD remain largely unknown, we performed a genome-wide scan for TPOAbs in 18,297 individuals, with replication in 8,990 individuals. Significant associations were detected with variants at TPO, ATXN2, BACH2, MAGI3, and KALRN. Individuals carrying multiple risk variants also had a higher risk of increased thyroid-stimulating hormone levels (including subclinical and overt hypothyroidism), and a decreased risk of goiter. The MAGI3 and BACH2 variants were associated with an increased risk of hyperthyroidism, and the MAGI3 variant was also associated with an increased risk of hypothyroidism. This first genome-wide scan for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease. With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. These results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which individuals are particularly at risk of developing clinical thyroid dysfunction.

Programmed cell death, or apoptosis, is inhibited by the antiapoptotic oncogene, Bcl-2, and is mediated by a cascade of aspartate-specific cysteine proteases, or caspases, related to interleukin 1-beta converting enzyme. Depending on cell type, apoptosis can be induced by treatment with thapsigargin (TG); a selective inhibitor of the endoplasmic reticulum-associated calcium-ATPase. The role of caspases in mediating TG-induced apoptosis was investigated in the Bcl-2-negative human breast cancer cell line, MDA-MB-468. Apoptosis developed in MDA-MB-468 cells over a period of 24-72 h following treatment with 100 nM TG, and was prevented by Bcl-2 overexpression. TG-induced apoptosis was associated with activation of caspase-3 and was inhibited by stable expression of the baculovirus p35 protein, an inhibitor of caspase activity. Also, TG-induced apoptosis was inhibited by treating cells with Z-VAD-fmk, a cell-permeable fluoromethylketone inhibitor of caspases. These findings indicate that TG-induced apoptosis of MDA-MB-468 breast cancer cells is subject to inhibition by Bcl-2 and is mediated by caspase activity. This model system should be useful for further investigation directed toward understanding the role of calcium in signaling apoptosis, and its relationship to Bcl-2 and the caspase proteolytic cascade.

Contains fulltext : 110933.pdf (Publisher’s version ) (Closed access) To search for sequence variants conferring risk of nonmedullary thyroid cancer, we focused our analysis on 22 SNPs with a P < 5 x 10(-8) in a genome-wide association study on levels of thyroid stimulating hormone (TSH) in 27,758 Icelanders. Of those, rs965513 has previously been shown to associate with thyroid cancer. The remaining 21 SNPs were genotyped in 561 Icelandic individuals with thyroid cancer (cases) and up to 40,013 controls. Variants suggestively associated with thyroid cancer (P < 0.05) were genotyped in an additional 595 non-Icelandic cases and 2,604 controls. After combining the results, three variants were shown to associate with thyroid cancer: rs966423 on 2q35 (OR = 1.34; P(combined) = 1.3 x 10(-9)), rs2439302 on 8p12 (OR = 1.36; P(combined) = 2.0 x 10(-9)) and rs116909374 on 14q13.3 (OR = 2.09; P(combined) = 4.6 x 10(-11)), a region previously reported to contain an uncorrelated variant conferring risk of thyroid cancer. A strong association (P = 9.1 x 10(-91)) was observed between rs2439302 on 8p12 and expression of NRG1, which encodes the signaling protein neuregulin 1, in blood. 01 maart 2012

Small nuclear RNAs (snRNAs) are essential factors in messenger RNA splicing. By means of homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays showed that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns were found to be poorly spliced in MOPD I patient fibroblast cells. The introduction of wild-type U4atac snRNA into MOPD I cells enhanced U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.

The strong genetic predisposition to papillary thyroid carcinoma (PTC) might be due to a combination of low-penetrance susceptibility variants. Thus, the research into gene variants involved in the increase of susceptibility to PTC is a relevant field of investigation. The gene coding for the receptor-type tyrosine phosphatase PTPRJ has been proposed as a cancer susceptibility gene, and its role as a tumor suppressor gene is well established in thyroid carcinogenesis. In this study, we want to ascertain the role of PTPRJ genotype in the risk for PTC. We performed a case–control study in which we determined the PTPRJ genotype for the non-synonymous Gln276Pro and Asp872Glu polymorphisms by PCR amplification and sequencing. We calculated allele and genotype frequencies for the considered polymorphisms of PTPRJ in a total sample of 299 cases (PTC patients) and 339 controls (healthy subjects) selected from Caucasian populations. We observed a significantly higher frequency of homozygotes for the Asp872 allele in the group of PTC patients than in the control group (odds ratio=1.61, 95% confidence interval 1.15–2.25, P=0.0053). We observed a non-significant increased frequency of homozygotes for Gln276Pro polymorphism in PTC cases in two distinct Caucasian populations. Therefore, the results reported here show that the homozygous genotype for Asp872 of PTPRJ is associated with an increased risk to develop PTC.

In order to search for sequence variants conferring risk of thyroid cancer we conducted a genome-wide association study in 192 and 37,196 Icelandic cases and controls, respectively, followed by a replication study in individuals of European descent. Here we show that two common variants, located on 9q22.33 and 14q13.3, are associated with the disease. Overall, the strongest association signals were observed for rs965513 on 9q22.33 (OR = 1.75; P = 1.7 x 10(-27)) and rs944289 on 14q13.3 (OR = 1.37; P = 2.0 x 10(-9)). The gene nearest to the 9q22.33 locus is FOXE1 (TTF2) and NKX2-1 (TTF1) is among the genes located at the 14q13.3 locus. Both variants contribute to an increased risk of both papillary and follicular thyroid cancer. Approximately 3.7% of individuals are homozygous for both variants, and their estimated risk of thyroid cancer is 5.7-fold greater than that of noncarriers. In a study on a large sample set from the general population, both risk alleles are associated with low concentrations of thyroid stimulating hormone (TSH), and the 9q22.33 allele is associated with low concentration of thyroxin (T(4)) and high concentration of triiodothyronine (T(3)).

Heritable allelic variation in gene expression may contribute to sporadic and familial disease, but is relatively unexplored. Papillary thyroid cancer (PTC) is characterized by strong heritability but predisposing genes have not been detected. Here we tested the hypothesis that inherited variation in allelic expression occurs in thyroid tissue and so might contribute to disease. We studied genes with a role in thyroid function, signaling, and/or tumorigenesis (PRKAR1A, DUOX1, IP3RI, ARGBP2, TPO, TG, and PEG10). We screened thyroid tissues from controls and patients with thyroid disease and lymphoblastoid cell lines from 40 healthy individuals. We demonstrated the robustness of the technique, a fluorescent dideoxy terminator-based method distinguishing the mRNA products of alleles in individuals who are heterozygous for a polymorphism in the transcript. We confirmed that the PEG10 gene transcript was derived only from one allele because of genetic imprinting. Three other genes, DUOX1, TPO, and ARGBP2 showed allelic variation in thyroid tissue and/or lymphoblastoid cells. Benign and malignant thyroid tissue from the same patient always gave concordant results. DUOX1 variation in expression was seen in 3 of 13 patients with PTC and 6 of 27 patients with benign disease. Further studies are needed to determine the significance of these and other allelic variations.

Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C) in a large pedigree displaying non-medullary thyroid carcinoma (NMTC). This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA) is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.

Gene expression analysis of glucocorticoid-induced apoptosis by a gene microarray technique