Pathogenic variations in MAML2 and MAMLD1 contribute to congenital hypothyroidism due to dyshormonogenesis by regulating the Notch signalling pathway.

BACKGROUND: In several countries, thyroid dyshormonogenesis is more common than thyroid dysgenesis in patients with congenital hypothyroidism (CH). However, known pathogenic genes are limited to those directly involved in hormone biosynthesis. The aetiology and pathogenesis of thyroid dyshormonogenesis remain unknown in many patients. METHODS: To identify additional candidate pathogenetic genes, we performed next-generation sequencing in 538 patients with CH and then confirmed the functions of the identified genes in vitro using HEK293T and Nthy-ori 3.1 cells, and in vivo using zebrafish and mouse model organisms. RESULTS: We identified one pathogenic MAML2 variant and two pathogenic MAMLD1 variants that downregulated canonical Notch signalling in three patients with CH. Zebrafish and mice treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester, a γ-secretase inhibitor exhibited clinical manifestations of hypothyroidism and thyroid dyshormonogenesis. Through organoid culture of primary mouse thyroid cells and transcriptome sequencing, we demonstrated that Notch signalling within thyroid cells directly affects thyroid hormone biosynthesis rather than follicular formation. Additionally, these three variants blocked the expression of genes associated with thyroid hormone biosynthesis, which was restored by HES1 expression. The MAML2 variant exerted a dominant-negative effect on both the canonical pathway and thyroid hormone biosynthesis. MAMLD1 also regulated hormone biosynthesis through the expression of HES3, the target gene of the non-canonical pathway. CONCLUSIONS: This study identified three mastermind-like family gene variants in CH and revealed that both canonical and non-canonical Notch signalling affected thyroid hormone biosynthesis.

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.

Contactin 6, A Novel Causative Gene for Congenital Hypothyroidism, Mediates Thyroid Hormone Biosynthesis Through Notch Signaling.

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.

Genetic screening and functional analysis of TPO variants in Chinese patients with congenital hypothyroidism.

INTRODUCTION: Congenital hypothyroidism (CH), the most common neonatal endocrine disorder world-wide, 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.

Erectile dysfunction in patients with traumatic urethral strictures treated with anastomotic urethroplasty: a single-factor analysis.

INTRODUCTION: To investigate factors correlated with erectile dysfunction (ED) in patients with traumatic urethral strictures undergoing end-to-end anastomotic urethroplasty (AU). MATERIALS AND METHODS: Between January 2010 and January 2011, 41 patients with urethral strictures resulting from pelvic fracture urethral distraction defects underwent end-to-end AU. The abridged International Index of Erectile Function (IIEF-5) was used to subjectively assess erectile function at admission and 2 weeks postoperatively. RESULTS: Pre- and post-injury IIEF-5 scores differed significantly (23.54 ± 1.45 versus 10.02 ± 3.57; p < 0.0001), but pre and postoperative scores did not (10.02 ± 3.57 versus 9.29 ± 4.14; p = 0.1560). Erectile function declined in all patients after injury and was postoperatively unchanged in 56.10%. Pre- and post-injury scores differed significantly in all ages, stricture location and length groups, but did not change postoperatively. Urethral injury resulted in varying degrees of ED. IIEF-5 scores declined significantly postoperatively in patients with mild/mild-moderate ED (13.86 ± 1.88 versus 11.43 ± 3.37; p = 0.0202), but were unchanged in patients with moderate/severe ED. Vascular ED was predominant (63.41%), and erectile function was better in patients with non-vascular ED than in those with arterial/venous ED (15.50 ± 2.08 versus 11.00 ± 2.35, 8.67 ± 3.21; p = 0.0037, p = 0.0183). IIEF-5 scores decreased significantly in patients with non-vascular ED postoperatively (15.50 ± 2.08 versus 10.00 ± 3.83; p = 0.0132), but were unchanged in patients with arterial/venous ED. CONCLUSION: Urethral trauma seriously affects erectile function, but subsequent end-to-end AU for urethral strictures has little impact.

H-/H∞ fault detection observer design for fractional-order singular systems in finite frequency domains.

This paper investigates the robust fault detection observer design problem in finite frequency domains for fractional-order singular systems. An H-/H∞ fault detection observer for fractional-order singular systems is designed to meet the system admissibility, disturbance robustness and fault sensitivity indices in finite frequency domains simultaneously. Four theorems and four corollaries about the system admissibility and the performance index H-/H∞ in different frequency ranges are given, and then the sufficient conditions are obtained in terms of linear matrix inequalities. Finally, two numerical examples are given to verify the validity of the presented methods.

Identification and Validation of Gene Expression Patterns in Cystitis Glandularis Patients and Controls.

Our aim was to investigate differences in gene expression in bladder tissues between cystitis glandularis (CG) patients and healthy controls. Subsequent RNA was isolated from urinary bladder samples from CG patients and healthy controls, followed by RNA sequencing analysis. There were 4263 differentially expressed genes in urinary bladder between CG patients and controls, and 8 genes were verified with real-time PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) analysis. Gene set enrichment analysis (GSEA) revealed that 25 signaling pathways were upregulated in CG patients, and 17 signaling pathways were found upregulated in healthy controls. The mRNA expression levels of the indicated genes, including CCND1, CCNA1, EGFR, AR, CX3CL1, CXCL6, and CXCL1, were significantly increased in urinary bladder from CG and bladder cancer (BC) patients compared with healthy controls, while TP53 was decreased. CX3CL1, CXCL6, and CXCL1 concentrations in peripheral blood from CG and BC patients were significantly increased compared with healthy controls. The protein expression levels of CCND1, EGFR, and AR were significantly increased in urinary bladder from CG and BC patients compared with healthy controls. In conclusion, the gene expression profile of CG patients has established a foundation to study the gene mechanism of CG and BC progression.