TAF1 is needed for the proliferation and maturation of thyroid follicle cells via Notch signaling.

Thyroid dysgenesis (TD) is the common pathogenic mechanism of congenital hypothyroidism (CH). In addition, known pathogenic genes are limited to those that are directly involved in thyroid development. To identify additional candidate pathogenetic genes, we performed forward genetic screening for TD in zebrafish, followed by positional cloning. The candidate gene was confirmed in vitro using the Nthy-ori 3.1 cell line and in vivo using a zebrafish model organism. We obtained a novel zebrafish line with thyroid dysgenesis and identified the candidate pathogenetic mutation TATA-box binding protein associated Factor 1 (taf1) by positional cloning. Further molecular studies revealed that taf1 was needed for the proliferation of thyroid follicular cells by binding to the NOTCH1 promoter region. Knockdown of TAF1 impaired the proliferation and maturation of thyroid cells, thereby leading to thyroid dysplasia. This study showed that TAF1 promoted Notch signaling and that this association played a pivotal role in thyroid development.NEW & NOTEWORTHY In our study, we obtained a novel zebrafish line with thyroid dysgenesis (TD) and identified the candidate pathogenetic mutation TATA-box binding protein associated Factor 1 (taf1). Further researches revealed that taf1 was required for thyroid follicular cells by binding to the NOTCH1 promoter region. Our findings revealed a novel role of TAF1 in thyroid morphogenesis.

Deficiency of the HGF/Met pathway leads to thyroid dysgenesis by impeding late thyroid expansion.

The mechanisms of bifurcation, a key step in thyroid development, are largely unknown. Here we find three zebrafish lines from a forward genetic screening with similar thyroid dysgenesis phenotypes and identify a stop-gain mutation in hgfa and two missense mutations in met by positional cloning from these zebrafish lines. The elongation of the thyroid primordium along the pharyngeal midline was dramatically disrupted in these zebrafish lines carrying a mutation in hgfa or met. Further studies show that MAPK inhibitor U0126 could mimic thyroid dysgenesis in zebrafish, and the phenotypes are rescued by overexpression of constitutively active MEK or Snail, downstream molecules of the HGF/Met pathway, in thyrocytes. Moreover, HGF promotes thyrocyte migration, which is probably mediated by downregulation of E-cadherin expression. The delayed bifurcation of the thyroid primordium is also observed in thyroid-specific Met knockout mice. Together, our findings reveal that HGF/Met is indispensable for the bifurcation of the thyroid primordium during thyroid development mediated by downregulation of E-cadherin in thyrocytes via MAPK-snail pathway.

TSHR Variant Screening and Phenotype Analysis in 367 Chinese Patients With Congenital Hypothyroidism.

Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype-phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions: We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.

ALYREF-mediated RNA 5-Methylcytosine modification Promotes Hepatocellular Carcinoma Progression Via Stabilizing EGFR mRNA and pSTAT3 activation.

5-Methylcytosine (m5C) is one of the most ubiquitous modifications of mRNA and contributes to cancer pathogenesis. Aly/REF export factor (ALYREF), an m5C reader, is associated with the prognosis of liver hepatocellular carcinoma (LIHC). However, the effects of ALYREF on the progression of LIHC and the underlying molecular mechanisms remains elusive. Through an analysis of an online database and 3 independent LIHC cohorts, we found that ALYREF was markedly elevated in human liver cancer tissues and was significantly correlated with LIHC clinicopathological parameters, including Ki67+ cell rate, high-grade TNM stage, and poor prognosis. Several experiments were conducted to investigate the molecular basis and functional role of ALYREF-related progression in this study. ALYREF could enhance LIHC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and tumor formation in vivo. Mechanistically, ALYREF promoted the progression of human LIHC through EGFR pathways. Furthermore, ALYREF could directly bind to the m5C modification site of EGFR 3' untranslated region (3' UTR) to stabilize EGFR mRNA. Collectively, ALYREF played a crucial oncogenic role in LIHC via the stabilization of EGFR mRNA and subsequent activation of the STAT3 signaling pathway. Our results may help to elucidate the potential mechanisms of ALYREF-induced m5C modification in the progression of human LIHC.

Myeloid cells interact with a subset of thyrocytes to promote their migration and follicle formation through NF-κB.

The pathogenesis of thyroid dysgenesis (TD) is not well understood. Here, using a combination of single-cell RNA and spatial transcriptome sequencing, we identify a subgroup of NF-κB-activated thyrocytes located at the center of thyroid tissues in postnatal mice, which maintained a partially mesenchymal phenotype. These cells actively protruded out of the thyroid primordium and generated new follicles in zebrafish embryos through continuous tracing. Suppressing NF-κB signaling affected thyrocyte migration and follicle formation, leading to a TD-like phenotype in both mice and zebrafish. Interestingly, during thyroid folliculogenesis, myeloid cells played a crucial role in promoting thyrocyte migration by maintaining close contact and secreting TNF-α. We found that cebpa mutant zebrafish, in which all myeloid cells were depleted, exhibited thyrocyte migration defects. Taken together, our results suggest that myeloid-derived TNF-α-induced NF-κB activation plays a critical role in promoting the migration of vertebrate thyrocytes for follicle generation.

YTHDF3as a prognostic predictive biomarker of thyroid cancer and its correlation with immune infiltration.

PURPOSE: Thyroid cancer (TC) is one of the most common endocrine malignancies, and its morbidity continues to rise. N6-methyladenosine (m6A) RNA methylation, an epigenetic modification, is an important regulator of gene expression in TC. Therefore, it's worth finding the characteristics and predictive value of the m6A RNA methylation regulators in thyroid cancer (TC). METHOD: RNA-seq data of TC was downloaded from the Cancer Genome Atlas (TCGA) database to screen out the differential expressed regulators. The absolute contraction selection operator (Lasso) Cox regression was used to construct the risk model of m6A methylation regulators. The predictive value of the risk scoring model was evaluated by Kaplan Meier (K-M) analysis and receiver operating characteristic (ROC) curves. The underlying mechanism of m6A methylation regulators in TC was predicted by gene set enrichment analysis (GSEA). Further validation was performed by using immunohistochemistry (IHC) and q-PCR. The correlation between risk-related gene and immune infiltration was evaluated by Tumour Immune Estimation Resource (TIMER). RESULTS: IGF2BP2, YTHDF1 and YTHDF3 were screened out as strong independent prognostic factors of TC. Then a risk score model was established to further screen the predictors. Finally, according to the results of overall survival (OS) and clinical characteristics of TC, YTHDF3 was screened out as a potential predictor. Meanwhile, IHC and qPCR confirmed that YTHDF3 was expressed differential in TC. The expression of YTHDF3 was positively associated with the infiltration level of CD4+ T cells and macrophages. It was strongly correlated with a variety of immune markers in TC. CONCLUSION: We confirmed that YTHDF3 can be used as a potential prognostic biomarker of TC. It not only plays a decisive role in the initiation and development of TC, but also provides a new perspective for understanding the modification of m6A RNA in TC.

Influence of two anti-tumor drugs, pazopanib, and axitinib, on the development and thyroid-axis of zebrafish (Danio rerio) embryos/larvae.

Introduction: In recent years, the potential toxicities of different pharmaceuticals toward the thyroid system have received increasing attention. In this study, we aim to evaluate the toxic effects of pazopanib and axitinib, two anti-tumor drugs with widespread clinical use, on thyroid function in the zebrafish model. Methods: We measured levels of thyroid-related hormones using the commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit. Whole-mount in situ hybridization (WISH) analysis was employed to detect target gene expression changes. Morphology of the thyroid were evaluated by using transgenic Tg (tg: EGFP) fish line under a confocal microscope. The relative mRNA expression of key genes was verified through quantitative real-time polymerase chain reaction (RT‒qPCR). The size and number of the follicles was quantified whereby Hematoxylin-Eosin (H & E) staining under a light microscope. Results: The results revealed that fertilized zebrafish embryos were incubated in pazopanib or axitinib for 96 hours, development and survival were significantly affected, which was accompanied by significant disturbances in thyroid endocrine system (e.g., increased thyroid-stimulating hormone (TSH) content and decreased triiodothyronine (T3) and thyroxine (T4) content, as well as transcription changes of genes associated with the hypothalamus-pituitary-thyroid (HPT) axis. Moreover, based on whole-mount in situ hybridization staining of tg and histopathological examination of zebrafish embryos treated with pazopanib and axitinib, we observed a significantly abnormal development of thyroid follicles in the Tg (tg: EGFP) zebrafish transgenic line. Conclusion: Collectively, these findings indicate that pazopanib and axitinib may have toxic effects on thyroid development and function, at least partially, by influencing the regulation of the HPT axis. Thus, we believe that the potential thyroid toxicities of pazopanib and axitinib in their clinical applications should receive greater attention.

Identification of SERPINA1 promoting better prognosis in papillary thyroid carcinoma along with Hashimoto's thyroiditis through WGCNA analysis.

Background: Hashimoto's thyroiditis (HT) is an autoimmune thyroid disease. Papillary thyroid carcinoma (PTC) is the most common endocrine cancer. In recent years the rate of coexistence between PTC and HT has increased but the relationship between them remains unclear, meaning it is necessary to find potential biomarkers for PTC coexistence with HT to predict its potential pathways. Method: A co-expression network was constructed using the weighted gene co-expression network analysis (WGCNA) in the R package. The modules of PTC associated with HT (PTC-W) were identified from the GSE138198 dataset. Protein-protein interaction network (PPI) was used to screen the hub genes. Immunohistochemical (IHC) analysis was performed to validate the expression of the hub genes in tissues. Clinical data from The Cancer Genome Atlas (TCGA) datasets were used to analyse the prognosis of the hub genes. Gene set enrichment analysis (GSEA) was used to screen potential pathways of PTC-W. Result: The MEbrown module representing the most significant module, with 958 differentially expressed genes (DEGs), was screened in PTC-W, based on WGCNA analysis. Through PPI, SERPINA1 was identified as a hub gene. Immunostaining validated that SERPINA1 was highly expressed in PTC-W. Moreover, PTC-W expressing SERPINA1 exhibits a better prognosis than PTC without HT (PTC-WO). Conclusion: Our study demonstrates that SERPINA1 promotes the occurrence of PTC-W, and its prognosis is better than PTC-WO. SERPINA1 promotes a better prognosis for PTC-W, possibly through a tumour inhibition signalling pathway.

The isl2a transcription factor regulates pituitary development in zebrafish.

Background: ISL LIM homeobox 2, also known as insulin gene enhancer protein ISL-2 (ISL2), is a transcription factor gene that participates in a wide range of developmental events. However, the role of ISL2 in the hypothalamus-pituitary-thyroid axis is largely unknown. In the present study, we characterized the expression patterns of ISL2 and revealed its regulative role during embryogenesis using zebrafish. Methods: We used the CRISPR/Cas9 system to successfully establish homozygous ISL2-orthologue (isl2a and isl2b) knockout zebrafish. Moreover, we utilized these knockout zebrafish to analyze the pituitary and thyroid phenotypes in vivo. For further molecular characterization, in situ hybridization and immunofluorescence were performed. Results: The isl2a mutant zebrafish presented with thyroid hypoplasia, reduced whole-body levels of thyroid hormones, increased early mortality, gender imbalance, and morphological retardation during maturity. Additionally, thyrotropes, a pituitary cell type, was notably decreased during development. Importantly, the transcriptional levels of pituitary-thyroid axis hormones-encoding genes, such as tshba, cga, and tg, were significantly decreased in isl2a mutants. Finally, the thyroid dysplasia in isl2a mutant larvae may be attributed to a reduction in proliferation rather than changes in apoptosis. Conclusions: In summary, isl2a regulates the transcriptional levels of marker genes in hypothalamus-pituitary-thyroid axis, and isl2a knockout causing low thyroid hormone levels in zebrafish. Thus, isl2a identified by the present study, is a novel regulator for pituitary cell differentiation in zebrafish, resulting in thyroid gland hypoplasia and phenotypes of hypothyroidism.

In-frame deletion of SMC5 related with the phenotype of primordial dwarfism, chromosomal instability and insulin resistance.

BACKGROUND: SMC5/6 complex plays a vital role in maintaining genome stability, yet the relationship with human diseases has not been described. METHODS: SMC5 variation was identified through whole-exome sequencing (WES) and verified by Sanger sequencing. Immunoprecipitation, cytogenetic analysis, fluorescence activated cell sorting (FACS) and electron microscopy were used to elucidate the cellular consequences of patient's cells. smc5 knockout (KO) zebrafish and Smc5K371del knock-in mouse models were generated by CRISPR-Cas9. RNA-seq, quantitative real-time PCR (qPCR), western blot, microquantitative computed tomography (microCT) and histology were used to explore phenotypic characteristics and potential mechanisms of the animal models. The effects of Smc5 knockdown on mitotic clonal expansion (MCE) during adipogenesis were investigated through Oil Red O staining, proliferation and apoptosis assays in vitro. RESULTS: We identified a homozygous in-frame deletion of Arg372 in SMC5, one of the core subunits of the SMC5/6 complex, from an adult patient with microcephalic primordial dwarfism, chromosomal instability and insulin resistance. SMC5 mutation disrupted its interaction with its interacting protein NSMCE2, leading to defects in DNA repair and chromosomal instability in patient fibroblasts. Smc5 KO zebrafish showed microcephaly, short length and disturbed glucose metabolism. Smc5 depletion triggers a p53-related apoptosis, as concomitant deletion of the p53 rescued growth defects phenotype in zebrafish. An smc5K371del knock-in mouse model exhibited high mortality, severe growth restriction and fat loss. In 3T3-L1 cells, the knockdown of smc5 results in impaired MCE, a crucial step in adipogenesis. This finding implies that defective cell survival and differentiation is an important mechanism linking growth disorders and metabolic homeostasis imbalance.

Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis.

The extracellular matrix (ECM) is made up of noncellular components that have special properties for influencing cell behavior and tissue structure. Small leucine-rich proteoglycans (SLRPs) are nonfibrillar ECM components that serve as structural scaffolds and signaling molecules. osteoglycin (OGN), a class III SLRP, is a ubiquitous ECM component that not only helps to organize the extracellular matrix but also regulates a number of important biological processes. As a glycosylated protein in the ECM, OGN was originally considered to be involved in fiber assembly and was reported to have a connection with fibrosis. In addition to these functions, OGN is found in a variety of cancer tissues and is implicated in cellular processes linked to tumorigenesis, including cell proliferation, invasion, metastasis, and epithelial-mesenchymal transition (EMT). In this review, we summarize the structure and functions of OGN as well as its biological and clinical importance in the context of fibrotic illness and tumorigenesis. This review aims to improve our understanding of OGN and provide some new strategies for the treatment of fibrosis and cancer.

The influence of sunitinib and sorafenib, two tyrosine kinase inhibitors, on development and thyroid system in zebrafish larvae.

Recently, the potential toxic effects of various pharmaceuticals on the thyroid endocrine system have raised considerable concerns. In this study, we evaluated the adverse effects of sorafenib and sunitinib, two widely used anti-tumor drugs, on the developmental toxicities and thyroid endocrine disruption by using zebrafish (Danio rerio) model. Zebrafish embryos/larvae were exposed to different contentions (0, 10, 50 and 100 nM) of sorafenib and sunitinib for 96 hpf. The results revealed that waterborne exposure to sorafenib and sunitinib exhibited remarkable toxic effects on the survival and development in zebrafish embryos/larvae, which was accompanied by obvious disturbances of thyroid endocrine system (e.g., decreased T3 and T4 content, increased TSH content) and genes' transcription changes within the hypothalamus-pituitary-thyroid (HPT) axis. In addition, we verified a strikingly abnormal thyroid gland organogenesis in zebrafish larvae in response to sorafenib and sunitinib, by assessing the development of thyroid follicles using the WISH staining of tg, the Tg (tg:GFP) zebrafish transgenic line, and histopathological analysis. Taken together, our results indicated sorafenib and sunitinib exposure could induce obvious developmental toxicities and thyroid function disruption in zebrafish embryos/larvae, which might involve a regulatory mechanism, at least in part, by destroying the thyroid follicle structure, and by disturbing the balance of the HPT axis.

Uterus globulin associated protein 1 (UGRP1) binds podoplanin (PDPN) to promote a novel inflammation pathway during Streptococcus pneumoniae infection.

BACKGROUND: Streptococcus pneumoniae is the major cause of life-threatening infections. Toll-like receptors (TLRs) and NOD-like receptors (NLRs) could recognise S. pneumoniae and regulate the production of pro-inflammatory cytokines. UGRP1, highly expressed in lung, is predominantly secreted in airways. However, the function of UGRP1 in pneumonia is mainly unknown. METHODS AND RESULTS: We showed that upon TLR2/TLR4/NOD2 agonists stimulation or S. pneumoniae infection, treatment with UGRP1 could promote phosphorylation of p65 and enhance IL-6, IL-1ß and TNFα production in macrophages. We further elucidated that after binding with cell-surface receptor PDPN, UGRP1 could activate RhoA to enhance interaction of IKKγ and IKKß, which slightly activated NF-κB to improve expression of TLR2, MyD88, NOD2 and NLRP3. Deletion of UGRP1 or blocking UGRP1 interaction with PDPN protected mice against S. pneumoniae-induced severe pneumococcal pneumonia, and activating RhoA with agonist in UGRP1-deficient mice restored the reduced IL-6 production. CONCLUSION: We demonstrated that UGRP1-PDPN-RhoA signaling could activate NF-κB to promote expression of TLR2, MyD88, NOD2 and NLRP3, which enhanced inflammatory cytokines secretion during S. pneumoniae infection. Antibodies, which could interrupt interaction of UGRP1 and PDPN, are potential therapeutics against S. pneumoniae.

Detection of BRAF V600E in Fine-Needle Aspiration Samples of Thyroid Nodules by Droplet Digital PCR.

Background: BRAF exon 15 p.V600E (BRAF V600E) mutation has been established as an important molecular marker for papillary thyroid carcinoma diagnosis by ultrasound-guided fine-needle aspiration biopsy (FNAB). Sanger sequencing is the gold standard for detecting BRAF V600E mutations but fails to identify low-frequency mutations. However, droplet digital PCR (ddPCR) is a popular new method for detecting low-frequency mutations. Here, we compare the efficiency of droplet digital PCR (ddPCR) and Sanger sequencing for detection of the BRAF V600E mutation in thyroid fine-needle aspiration (FNA) samples. Methods: Thyroid fine-needle aspiration samples from 278 patients with 310 thyroid nodules were collected. Sanger sequencing and ddPCR were conducted to detect the BRAF V600E mutation. Results: The BRAF V600E mutation was found in 94 nodules (30.32%) by ddPCR and 40 nodules (12.90%) by Sanger sequencing in 310 FNA samples. A total of 119 nodules were confirmed PTC by postsurgical pathology. Among which the BRAF mutation was found in 80 (67.23%) nodules by ddPCR and 31 (26.05%) by Sanger sequencing. All nodules carrying the mutation detected by Sanger sequencing (SS+) were verified by ddPCR (ddPCR+). Also, all nodules with no mutation detected by ddPCR were interpreted as wild-type by Sanger sequencing (SS-). In addition. Almost all SS+/ddPCR + nodules (95.00%; 38/40) and SS-/ddPCR + nodules (100.00%; 54/54) displayed a BRAF mutation rate of >5% and <15%, respectively, indicating easy misdetection by Sanger sequencing when the mutation rate is between 5 and 15%. Conclusion: ddPCR has higher sensitivity than Sanger sequencing and we propose ddPCR as a supplement to Sanger sequencing in molecular testing of BRAF using FNAB samples.

Compelling Evidence Linking CD40 Gene With Graves' Disease in the Chinese Han Population.

Mutations in CD40 have been widely reported to be risk factors for Graves' disease (GD). The gene, along with its cognate ligand CD40L, may regulate pro-inflammatory and immune responses. Rs1883832, located at the -1 position of the Kozak sequence, is the most well-studied single nucleotide polymorphism (SNP) of CD40, and has been confirmed to predispose those with the alteration to GD, regardless of ethnicity. Our genome-wide association study (GWAS) indicated that several SNPs, including rs1883832 located within the vicinity of CD40 were associated with GD in the Han Chinese population. Aiming at identifying the most consequential SNP and its underlying pathogenic mechanism, we performed a two-stage refined study on 8,171 patients with GD and 7,906 controls, and found rs1883832 was the most significantly GD-associated SNP in the CD40 gene region (PCombined = 9.17×10-11, OR = 1.18). Through searching the cis-expression quantitative trait locus database and using quantitative RT-PCR, we further discovered that the rs1883832 genotype can influence CD40 gene transcription. Furthermore, we demonstrated that rs1883832 is a susceptibility locus for pTRAb+ GD patients. In conclusion, the current study provides robust evidence that rs1883832 can regulate CD40 gene expression and affect serum TRAb levels, which ultimately contributes to the development of GD.

The effect of radioiodine treatment on the characteristics of TRAb in Graves' disease.

BACKGROUND: Graves' disease (GD) is one of the most common autoimmune thyroid diseases (AITDs) in humans, and thyrotropin receptor antibody (TRAb) is a characterized autoantibody in GD. The use of radioactive iodine therapy (RAI) for GD treatment is increasing. OBJECTIVES: We studied the biological properties of TRAb and evaluated the effect of RAI therapy on TRAb in GD patients. METHODS: In total, 225 patients (22 onset GD patients without 131I therapy, 203 GD patients treated with 131I therapy) and 20 healthy individuals as normal controls were included in this study. Clinical assessments were performed, and we examined in vitro the biological properties of TRAb in the 22 onset GD patients and 20 controls as well as 84 GD patients with 131I therapy. RESULTS: Serum TRAb and thyroid peroxidase antibody (TPOAb) levels increased in the initial year of RAI treatment, and both antibodies decreased gradually after one year. After 5 years from radioiodine treatment, TRAb and TPOAb levels decreased in 88% and 65% of GD patients, respectively. The proportion of patients positive for thyroid-stimulatory antibody (TSAb) was significantly higher in the 7-12-month group, and thyroid-blocking antibody (TBAb) levels were elevated after one year in half of the patients who received 131I treatment. CONCLUSIONS: Treatment of GD patients with radioiodine increased TPOAb and TRAb (their main biological properties were TSAbs) within the first year after therapy, and the main biological properties of elevated TRAb were TBAbs after 1 year.

A five-gene panel refines differential diagnosis of thyroid nodules.

BACKGROUND: Molecular testing for oncogenic mutations in fine-needle aspiration has showed high predictive value in identifying malignant lesions from thyroid nodules with indeterminate cytology. METHODS: To figure out an efficient and economical gene panel for most medical institutions in China, we designed a five-gene panel including BRAF/NRAS/KRAS/HRAS/TERT genes and conducted a retrospective study to evaluate the role of this five-gene diagnostic panel in differential diagnosis of thyroid nodules. RESULTS: A total of 665 patients with 695 thyroid nodules were investigated in the current study. The fine-needle aspiration biopsy and surgically separated thyroid tissue specimens were harvested to test BRAF, TERT, NRAS, KRAS, and HRAS mutations. We identified 261 mutations in 665 patients, including 177 V600E mutations in BRAF. Three hundred and sixty-nine patients who underwent thyroid surgery after completion of the initial clinical and cytological evaluation were enrolled in the final analysis. The diagnostic sensitivity, specificity, and accuracy of the combination of FNAB cytology and five-gene detection were 74.7%, 93.8%, and 84.8%, respectively. BRAF V600E and five-gene panel could recognize 46.4% and 53.6% of papillary thyroid carcinoma in the patients with cytologically indeterminate nodules. CONCLUSION: The five-gene panel can effectively improve the sensitivity, negative predictive value, and accuracy of fine-needle aspiration biopsy cytology, especially in the patients with cytologically indeterminate nodules.

Exome sequencing identifies somatic mutations in novel driver genes in non-small cell lung cancer.

Lung cancer is the leading cause of cancer death worldwide and accounts for more than one-third of all newly diagnosed cancer cases in China. Therefore, it is of great clinical significance to explore new driver gene mutations in non-small-cell lung cancer (NSCLC). Using an initial bioinformatic analysis, we identified somatic gene mutations in 13 patients with NSCLC and confirmed these mutations by targeted sequencing in an extended validation group of 88 patients. Recurrent mutations were detected in UNC5D (7.9%), PREX1 (5.0%), HECW1 (4.0%), DACH1 (2.0%), and GPC5 (2.0%). A functional study was also performed in UNC5D mutants. Mutations in UNC5D promoted tumorigenesis by abolishing the tumor suppressor function of the encoded protein. Additionally, in ten patients with lung squamous cell carcinoma, we identified mutations in KEAP1/NFE2L2 that influenced the expression of target genes in vivo and in vitro. Overall, the results of our study expanded the known spectrum of driver mutations involved in the pathogenesis of NSCLC.

Uterus globulin associated protein 1 (UGRP1) is a potential marker of progression of Graves' disease into hypothyroidism.

Approximately 20% of Graves' disease (GD) patients may result eventually in hypothyroidism in their natural course. Uterus globulin-associated protein 1 (UGRP1) was associated with GD in our previous study. Here we investigated the role of UGRP1 in the development of autoimmune thyroid disease (AITD). The results showed that UGRP1 was expressed in the thyrocytes of most Hashimoto's thyroiditis (HT) patients and a proportion of GD patients (293 HT and 198 GD). The pathologic features of UGRP1-positive thyrocytes resembled "Hürthle cells", and were surrounded by infiltrated leukocytes. The positivity rate of TPOAb in UGRP1-positive GD patients was much higher than that in -negative GD patients. Moreover, UGRP1 was co-expressed with Fas and HLA-DR in the thyrocytes of AITD patients. We also found IL-1ß but not Th1 or Th2 cytokines was able to upregulate the expression of UGRP1. Our findings indicated that UGRP1 may be a novel marker in thyrocytes to predict GD patients who develop hypothyroidism.

Identification of three novel SRD5A2 mutations in Chinese patients with 5α-reductase 2 deficiency.

In this study, we investigated the genetics, clinical features, and therapeutic approach of 14 patients with 5α-reductase deficiency in China. Genotyping analysis was performed by direct sequencing of PCR products of the steroid 5α-reductase type 2 gene (SRD5A2). The 5α-reductase activities of three novel mutations were investigated by mutagenesis and an in vitro transfection assay. Most patients presented with a microphallus, variable degrees of hypospadias, and cryptorchidism. Eight of 14 patients (57.1%) were initially reared as females and changed their social gender from female to male after puberty. Nine mutations were identified in the 14 patients. p.G203S, p.Q6X, and p.R227Q were the most prevalent mutations. Three mutations (p.K35N, p.H162P, and p.Y136X) have not been reported previously. The nonsense mutation p.Y136X abolished enzymatic activity, whereas p.K35N and p.H162P retained partial enzymatic activity. Topical administration of dihydrotestosterone during infancy or early childhood combined with hypospadia repair surgery had good therapeutic results. In conclusion, we expand the mutation profile of SRD5A2 in the Chinese population. A rational clinical approach to this disorder requires early and accurate diagnosis, especially genetic diagnosis.