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Introduction: Congenital hypothyroidism (CH), the most common neonatal endocrine disorder worldwide, can be caused by variants in the thyroid peroxidase (TPO) gene. This study aimed to identify TPO variants in Chinese patients with CH, analyze their impact on TPO function, and establish relationships between TPO genotypes and clinical characteristics. Methods: A total of 328 patients with CH were screened for TPO variants by performing whole-exome sequencing. The function of the detected TPO variants was investigated via transfection assays in vitro. The pathogenic effect of five novel variants was further assessed in silico. Results: Among 328 patients with CH, 19 TPO variants, including six novel ones, were identified in 43 patients. Eighteen patients (5.5%) carried biallelic TPO variants. In vitro experiments showed that TPO activity was impaired to varying degrees in 17 variants. Furthermore, we determined that a residual TPO enzyme activity threshold of 15% may serve as a criterion for differentiating CH severity. Conclusions: According to our study, the prevalence of TPO variants among Chinese patients with CH was 13.1%. Five novel variants led to impaired TPO function by altering its structure or by affecting its expression or cellular localization, which should result in impaired thyroid hormone synthesis. [ABSTRACT FROM AUTHOR]

Background: Congenital hypothyroidism (CH) is the most common neonatal metabolic disorder. In patients with CH in China, thyroid dyshormonogenesis is more common than thyroid dysgenesis; however, the genetic causes of CH due to thyroid dyshormonogenesis remain largely unknown. Therefore, we aimed at identifying novel candidate causative genes for CH. Methods: To identify novel CH candidate genes, a total of 599 patients with CH were enrolled and next-generation sequencing was performed. The functions of the identified variants were confirmed using HEK293T and FTC-133 cell lines in vitro and in a mouse model organism in vivo. Results: Three pathogenic contactin 6 (CNTN6) variants were identified in two patients with CH. Pedigree analysis showed that CH caused by CNTN6 variants was inherited in an autosomal recessive pattern. The CNTN6 gene was highly expressed in the thyroid in humans and mice. Cntn6 knockout mice presented with thyroid dyshormonogenesis and CH due to the decreased expression of crucial genes for thyroid hormone biosynthesis (Slc5a5, Tpo, and Duox2). All three CNTN6 variants resulted in the blocking of the release of the Notch intracellular domain, which could not translocate into the nucleus, impaired NOTCH1 transcriptional activity, and decreased expression of SLC5A5, TPO, and DUOX2. Further, we found that DTX1 was required for CNTN6 to promote thyroid hormone biosynthesis through Notch signaling. Conclusions: This study demonstrated that CNTN6 is a novel causative gene for CH through the mediation of thyroid hormone biosynthesis via Notch signaling, which provides new insights into the genetic background and mechanisms involved in CH and thyroid dyshormonogenesis. [ABSTRACT FROM AUTHOR]

Objective: Congenital hypothyroidism (CH) is known to be due to thyroid dyshormonogenesis (DH), which is mostly inherited in an autosomal recessive inheritance pattern or thyroid dysgenesis (TD), whose inheritance pattern is controversial and whose molecular etiology remains poorly understood. Design and Methods: The variants in 37 candidate genes of CH, including 25 genes related to TD, were screened by targeted exon sequencing in 205 Chinese patients whose CH cannot be explained by biallelic variants in genes related to DH. The inheritance pattern of the genes was analyzed in family trios or quartets. Results: Of the 205 patients, 83 patients carried at least one variant in 19 genes related to TD, and 59 of those 83 patients harbored more than two variants in distinct candidate genes for CH. Biallelic or de novo variants in the genes related to TD in Chinese patients are rare. We also found nine probands carried only one heterozygous variant in the genes related to TD that were inherited from a euthyroid either paternal or maternal parent. These findings did not support the monogenic inheritance pattern of the genes related to TD in CH patients. Notably, in family trio or quartet analysis, of 36 patients carrying more than two variants in distinct genes, 24 patients carried these variants inherited from both their parents, which indicated that the oligogenic inheritance pattern of the genes related to TD should be considered in CH. Conclusions: Our study expanded the variant spectrum of the genes related to TD in Chinese CH patients. It is rare that CH in Chinese patients could be explained by monogenic germline variants in genes related to TD. The hypothesis of an oligogenic origin of the CH should be considered. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

DUOX2 is the most frequently mutated gene in patients with congenital hypothyroidism (CH) in China. However, no reliable genotype–phenotype relationship has been found in patients with DUOX2 mutations. In this study, DUOX2 mutations were screened in 266 CH patients, and the enzymatic activity of 89 DUOX2 variants was determined in vitro. Furthermore, the DUOX2 residual activity in 76 CH patients caused by DUOX2 biallelic mutations was calculated. The thyroid‐stimulating hormone (TSH) and free thyroxine (FT4) levels were found to be higher and lower in patients with DUOX2 residual activity ≤22%, respectively, compared to patients with residual enzymatic activity >22%. Moreover, we interpreted the pathogenicity of DUOX2 variants by applying the ACMG classification criteria with or without PS3/BS3 evidence. The results indicated that residual DUOX2 enzymatic activity was closely related to the clinical phenotypes of CH patients caused by DUOX2 biallelic mutations. These findings suggest that the residual enzymatic activity of 22% may be a cutoff value for estimating the severity of hypothyroidism in CH patients with biallelic DUOX2 mutations. Well‐established functional studies are useful and necessary to evaluate the pathogenicity of DUOX2 variants, improving the accuracy and scope of genetic consultations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (P Combined = 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. [ABSTRACT FROM AUTHOR]

Congenital hypothyroidism (CH) is a highly prevalent but treatable neonatal endocrine disorder. Thyroid dyshormonogenesis is the main cause of congenital hypothyroidism in Chinese CH patients, and DUOX2 is the most frequent mutated gene involved in H2O2 production. In humans, the primary sources for H2O2 production are DUOX1 and DUOX2 , while in zebrafish there is only a single orthologue for DUOX1 and DUOX2. In this study, duox mutant zebrafish were generated through knockdown duox by morpholino or knockout duox by CRISPR Cas9. The associated phenotypes were investigated and rescued by thyroxine (T4) treatment. Mutant zebrafish displayed hypothyroid phenotypes including growth retardation, goiter and, infertility. Homozygous mutants in adults also displayed extrathyroidal abnormal phenotypes, including lacking barbels, pigmentation defects, erythema in the opercular region, ragged fins, and delayed scales. All these abnormal phenotypes can be rescued by 10 nM T4 treatment. Strikingly, the fertility of zebrafish was dependent on thyroid hormone; T4 treatment should be continued and cannot be stopped over 2 weeks in hypothyroid zebrafish in order to achieve fertility. Thyroid hormones played a role in the developing and maturing of reproductive cells. Our work indicated that duox mutant zebrafish may provide a model for human congenital hypothyroidism. [ABSTRACT FROM AUTHOR]

Thyroid dysgenesis (TD) is a major cause of primary congenital hypothyroidism; however, the molecular mechanism underlying this process is unclear. Current knowledge regarding the morphogenesis of the thyroid gland and vascular anomalies affecting thyroid development is limited. To monitor the early stages of thyroid gland development, we generated double transgenic zebrafish embryos Tg(tg:mCherry/flk1:EGFP). We described the volume of the thyroid from 2 days postfertilization (dpf) to 5 dpf using 3D reconstruction images. We treated zebrafish embryos with the fibroblast growth factor (FGF) inhibitor PD166866 to better understand the impact of vascular defects on thyroid development and the effects of drug administration at specific time periods on different stages of thyroid development. The 3D reconstruction data revealed that the thyroid glands underwent significant transformation at critical time points. PD166866 treatment from 48 to 72 hours postfertilization (hpf) and from 72 to 96 hpf did not cause obvious reductions in thyroid volume but did result in observable abnormalities in thyroid morphology. The treatment also affected thyroid volume from 36 to 48 hpf, thus indicating that there are time‐point‐specific effects of drug administration during thyroid development. Three‐dimensional image reconstruction provides a comprehensive picture of thyroid anatomy and can be used to complement anatomical fluorescence information. The effects of an FGF pathway inhibitor on thyroid development were determined to be time‐point‐dependent. [ABSTRACT FROM AUTHOR]

Background and Objectives: Defects in the human sodium/iodide symporter (SLC5A5) gene have been reported to be one of the causes of congenital hypothyroidism (CH). We aimed to identify SLC5A5 mutations in Chinese patients with CH and to evaluate the function of the mutation. Methods: Two hundred and seventy-three patients with primary CH were screened for mutations in SLC5A5 using next-generation sequencing. We investigated the expression and cellular localization of the novel compound heterozygous mutation in SLC5A5. The functional activity of the mutants was further examined in vitro. Results: In 273 patients with CH, two previously undescribed pathogenic mutations p.Gly51AlafsTer45 (G51fs) and p.Gly421Arg (G421R) in a compound heterozygous state in SLC5A5 were identified in a pediatric patient. G51fs was located in the first intercellular loop connecting transmembrane segment I and II, whereas G421R was in the transmembrane segment (TMS) XI. G51fs and G421R resulted in a truncated NIS and reduced protein expression, respectively. In vitro experiments further showed that the normal function of iodine transport of sodium-iodide symporter (NIS) mutants was markedly impaired. Conclusion: The undescribed compound heterozygous mutation of SLC5A5 was discovered in a Chinese CH patient. The mutation led to significantly reduced NIS expression and impaired iodide transport function accompanied by the impaired location of the NIS on the plasma membrane. Our study thus provides further insights into the roles of SLC5A5 in CH pathogenesis. [ABSTRACT FROM AUTHOR]

Background: We aimed to examine the association of urinary iodine concentration with Hashimoto's thyroiditis (HT) risk, and particularly, to investigate whether the HT-related genetic variations might modify the effects of urinary iodine on HT in the Chinese Han population. Methods: We conducted a case–control study with 1723 Chinese (731 cases, 992 controls). The associations between urinary iodine concentration and HT risk were analyzed using logistic regression models. The effects of interactions between the genetic risk scores (GRSs) and urinary iodine on HT risk were assessed by including the respective interaction terms in the models. We also applied restricted cubic spline regression to estimate the possible nonlinear relationship. The multinomial logistic regression models were performed to determine the associations of urinary iodine with euthyroid-HT and hypothyroidism-HT. Results: After controlling for potential confounders, the odds of HT increased with increasing quartiles of urinary iodine concentration: adjusted odds ratios (ORs) and 95% confidence intervals [CIs] were 1.45 [1.06–1.99], 1.66 [1.17–2.34], and 2.07 [1.38–3.10] for the quartiles 2, 3, and 4, respectively, compared with the first quartile (p for trend <0.001). Multivariable restricted cubic spline regression analysis further demonstrated that there was a near-linear association between urinary iodine concentration and HT risk (p-overall <0.001; p-nonlinear = 0.074). However, we did not find significant interactions between urinary iodine and GRSs on the risk of HT (all p for interaction >0.05). Interestingly, we found that each increment of urinary iodine was associated with a more than twofold increase in the odds of hypothyroidism-HT (adjusted OR = 2.64 [CI = 1.73–4.05]), but not with euthyroid-HT (p > 0.05). Conclusions: Higher urinary iodine concentration was associated with increased risk of HT, and this association was near linear, indicating that increased urinary iodine has a continuous and graded impact on HT risk. Moreover, the iodine-HT association was not modified by genetic predisposition to HT. Interestingly, urinary iodine concentration was significantly associated with increased risk of hypothyroidism. [ABSTRACT FROM AUTHOR]

Background: With several susceptibility single nucleotide polymorphisms identified by case–control association studies, Graves' disease is one of the most common forms of autoimmune thyroid disease. In this study, we aimed to determine whether any observed differences in genetic associations are influenced by sex in Chinese Han populations. Methods: A total of 8,835 patients with Graves' disease and 9,936 sex‐matched healthy controls were enrolled in the study. Confirmed by a two‐staged association analysis, sex‐specific analyses among 20 Graves' disease susceptibility loci were conducted. Results: A significant sex‐gene interaction was detected primarily at rs5912838 on Xq21.1 between the GPR174 and ITM2A genes, whereby male Graves' disease patients possessed a significantly higher frequency of risk alleles than their female counterparts. Interestingly, compared to women, male patients with Graves' disease had a higher cumulative genetic risk and higher persistent thyroid stimulating hormone receptor antibody‐positive rate after receiving antithyroid drug therapy for at least 1 year. Conclusion: The findings of this study suggest the existence of one potential sex‐specific Graves' disease variant on Xq21.1. This could increase our understanding of the pivotal mechanism behind Graves' disease and ultimately aid in identifying possible therapeutic targets. [ABSTRACT FROM AUTHOR]

Background. A deficiency in steroid 5α-reductase type 2 is an autosomal recessive disorder. Affected individuals manifested ambiguous genitalia, which is caused by decreased dihydrotestosterone (DHT) synthesis in the fetus. Methods. We analyzed 25 patients with 5α-reductase deficiency in China. Seventeen of the 25 patients (68%) were initially raised as females. Sixteen patients changed their social gender from female to male after puberty. Results. Eighteen mutations were identified in these patients. p.Gly203Ser and p.Gln6 ∗ were found to be the most prevalent mutations. On the basis of the genotype of these patients, we divided them into different groups. There was no significant difference in hormone levels and external masculinization score (EMS) in patients with or without these prevalent mutations. Twelve common single-nucleotide polymorphisms (SNPs) near the p.Gln6 ∗ mutation were chosen for haplotype analysis. Three haplotypes were observed in 6 patients who had the p.Gln6 ∗ mutation (12 alleles). Conclusion. We analyzed mutations of the SRD5A2 gene in Chinese patients with 5α-reductase deficiency. Although hotspot mutations exist, no founder effect of prevalent mutations in the SRD5A2 gene was detected in the Chinese population. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Objective: We aimed to investigate the six susceptibility loci of GD identified from European population in Chinese Han population and further to estimate the genetic heterogeneity of them in stratification of our GD patients. Design: Dense mapping studies based on GWAS. Patients: A total of 1536 GD patients and 1516 controls in GWAS stage and 1994 GD patients and 2085 controls and 5033 GD patients and 5389 controls in two replication stages. Measurements: Based on our previous GWAS data, independently GD‐associated SNPs in each region were identified by TagSNP analysis and logistic regression analysis. The association of these SNPs was investigated in 1994 GD patients and 2085 controls, and then, the significantly associated SNPs (P < 0.05) were further genotyped in a second cohort including 5033 GD patients and 5389 controls. Results: After the first replication stage, four SNPs from three regions with Pfirst < 0.05 were further selected and genotyped in another independent cohort. The association of two SNPs with GD was confirmed in combined Chinese cohorts: rs12575636 at 11q21 (Pcombined = 7.55 × 10−11, OR = 1.27) and rs1881145 in TRIB2 at 2p25.1 (Pcombined = 5.59 × 10−8, OR = 1.14). Further study disclosed no significant difference for these SNPs between GD subsets. However, eQTL data revealed that SESN3 could be a potential susceptibility gene of GD in 11q21 region. Conclusions: Out of the six susceptibility loci of GD identified from European population, two risk loci were confirmed in a large Chinese Han population. There is variability in GD genetic susceptibility in different ethnic groups. SESN3 is a potential susceptible gene of GD in 11q21. [ABSTRACT FROM AUTHOR]

Intratumor heterogeneity (ITH) in non-small cell lung cancer (NSCLC) may account for resistance after a period of targeted therapies because drugs destroy only a portion of tumor cells. The recognition of ITH helps identify high-risk patients to make effective treatment decisions. However, ITH studies are confounded by interpatient heterogeneity in NSCLC and a large amount of passenger mutations. To address these issues, we recruited NSCLC patients carrying TP53 mutations and selected driver mutations within recurrently mutated genes in NSCLC. A total of 12-paired normal-tumor tissues were subjected to whole-genome/whole-exome sequencing. From these, 367 non-silent mutations were selected as driver mutations and deeply sequenced in 61 intratumoral microdissections. We identified a universal prevalence of heterogeneity in all 12 tumors, indicating branched evolution. Although TP53 mutations were observed in single biopsy of all 12 tumors, most tumors consist of both TP53 mutated and non-mutated cells in separate regions within the same tumor. This suggests the late molecular timing of the acquisition of TP53 mutations; therefore, the detection of TP53 mutations in a single biopsy may simply not reflect the early malignant potential. In addition, we identified regions of loss of heterozygosity surrounding TP53 and CDKN2A mutations in tumor 711, which also exhibited heterogeneity in different regional samples. Because the ITH of driver mutations likely has clinical consequences, further efforts are needed to limit the impact of ITH and to improve therapeutic efficiency, which will benefit NSCLC patients receiving targeted treatments. [ABSTRACT FROM AUTHOR]