STR mutations on chromosome 15q cause thyrotropin resistance by activating a primate-specific enhancer of MIR7-2/MIR1179.

Thyrotropin (TSH) is the master regulator of thyroid gland growth and function. Resistance to TSH (RTSH) describes conditions with reduced sensitivity to TSH. Dominantly inherited RTSH has been linked to a locus on chromosome 15q, but its genetic basis has remained elusive. Here we show that non-coding mutations in a (TTTG)4 short tandem repeat (STR) underlie dominantly inherited RTSH in all 82 affected participants from 12 unrelated families. The STR is contained in a primate-specific Alu retrotransposon with thyroid-specific cis-regulatory chromatin features. Fiber-seq and RNA-seq studies revealed that the mutant STR activates a thyroid-specific enhancer cluster, leading to haplotype-specific upregulation of the bicistronic MIR7-2/MIR1179 locus 35 kb downstream and overexpression of its microRNA products in the participants' thyrocytes. An imbalance in signaling pathways targeted by these micro-RNAs provides a working model for this cause of RTSH. This finding broadens our current knowledge of genetic defects altering pituitary-thyroid feedback regulation.

A Novel Pathogenic Variant in PAX8 Leads to Familial Congenital Hypothyroidism.

We report a 10-month-old girl with familial congenital hypothyroidism harboring a novel heterozygous pathogenic variant in the paired DNA-binding domain of PAX8 (NM_003466:c.110T>C:p.Leu37Pro). Genotype-phenotype correlation revealed complete penetrance of this PAX8 defect in this family, in which the affected father and half-brother carry the same mutation. This deleterious variant has not been reported in any of the available databases [MAFgnomAD = 0, dbSNP (-)], and the amino acid leucine at position 37 is highly conserved across species. Establishing the molecular diagnosis expands our knowledge on the cause of thyroid dysgenesis and provides a guide for counseling and early treatment.

Genetics of ovarian insufficiency and defects of folliculogenesis.

Primary ovarian insufficiency (POI) is determined by exhaustion of follicles in the ovaries, which leads to infertility before the age of 40 years. It is characterized by a strong familial and heterogeneous genetic background. Therefore, we will mainly discuss the genetic basis of POI in this review. We identified 107 genes related to POI etiology in mammals described by several independent groups. Thirty-four of these genes (AARS2, AIRE, ANTXR1, ATM, BMPR1B, CLPP, CYP17A1, CYP19A1, DCAF17, EIF2B, ERAL1, FANCA, FANCC, FMR1, FOXL2, GALT, GNAS, HARS2, HSD17B4, LARS2, LMNA, MGME1, NBN, PMM2, POLG, PREPL, RCBTB1, RECQL2/3/4, STAR, TWNK, and XRCC4/9) have been linked to syndromic POI and are mainly implicated in metabolism function and meiosis/DNA repair. In addition, the majority of genes associated with nonsyndromic POI, widely expanded by high-throughput techniques over the last decade, have been implicated in ovarian development and meiosis/DNA repair pathways (ATG7, ATG9, ANKRD31, BMP8B, BMP15, BMPR1A, BMPR1B, BMPR2, BNC1, BRCA2, CPEB1, C14ORF39, DAZL, DIAPH2, DMC1, ERCC6, FANCL, FANCM, FIGLA, FSHR, GATA4, GDF9, GJA4, HELQ, HSF2BP, HFM1, INSL3, LHCGR, LHX8, MCM8, MCM9, MEIOB, MSH4, MSH5, NANOS3, NOBOX, NOTCH2, NR5A1, NUP107, PGRMC1, POLR3H, PRDM1, PRDM9, PSMC3IP, SOHLH1, SOHLH2, SPIDR, STAG3, SYCE1, TP63, UBR2, WDR62, and XRCC2), whereas a few are related to metabolic functions (EIF4ENIF1, KHDRBS1, MRPS22, POLR2C). Some genes, such as STRA8, FOXO3A, KIT, KITL, WNT4, and FANCE, have been shown to cause ovarian insufficiency in rodents, but mutations in these genes have yet to be elucidated in women affected by POI. Lastly, some genes have been rarely implicated in its etiology (AMH, AMHR2, ERRC2, ESR1, INHA, LMN4, POF1B, POU5F1, REC8, SMC1B). Considering the heterogeneous genetic and familial background of this disorder, we hope that an overview of literature data would reinforce that genetic screening of those patients is worthwhile and helpful for better genetic counseling and patient management.

Genetics of Primary Ovarian Insufficiency in the Next-Generation Sequencing Era.

Primary ovarian insufficiency (POI) is characterized by amenorrhea, increased follicle-stimulating hormone (FSH) levels, and hypoestrogenism, leading to infertility before the age of 40 years. Elucidating the cause of POI is a key point for diagnosing and treating affected women. Here, we review the genetic etiology of POI, highlighting new genes identified in the last few years using next-generation sequencing (NGS) approaches. We searched the MEDLINE/PubMed, Cochrane, and Web of Science databases for articles published in or translated to English. Several genes were found to be associated with POI genetic etiology in humans and animal models (SPIDR, BMPR2, MSH4, MSH5, GJA4, FANCM, POLR2C, MRPS22, KHDRBS1, BNC1, WDR62, ATG7/ATG9, BRCA2, NOTCH2, POLR3H, and TP63). The heterogeneity of POI etiology has been revealed to be remarkable in the NGS era, and discoveries have indicated that meiosis and DNA repair play key roles in POI development.

New evidences on the regulation of SF-1 expression by POD1/TCF21 in adrenocortical tumor cells.

OBJECTIVES:: Transcription Factor 21 represses steroidogenic factor 1, a nuclear receptor required for gonadal development, sex determination and the regulation of adrenogonadal steroidogenesis. The aim of this study was to investigate whether silencing or overexpression of the gene Transcription Factor 21 could modulate the gene and protein expression of steroidogenic factor 1 in adrenocortical tumors. METHODS:: We analyzed the gene expression of steroidogenic factor 1 using qPCR after silencing endogenous Transcription Factor 21 in pediatric adrenal adenoma-T7 cells through small interfering RNA. In addition, using overexpression of Transcription Factor 21 in human adrenocortical carcinoma cells, we analyzed the protein expression of steroidogenic factor 1 using Western blotting. RESULTS:: Transcription Factor 21 knockdown increased the mRNA expression of steroidogenic factor 1 by 5.97-fold in pediatric adrenal adenoma-T7 cells. Additionally, Transcription Factor 21 overexpression inhibited the protein expression of steroidogenic factor 1 by 0.41-fold and 0.64-fold in two different adult adrenocortical carcinoma cell cultures, H295R and T36, respectively. CONCLUSIONS:: Transcription Factor 21 is downregulated in adrenocortical carcinoma cells. Taken together, these findings support the hypothesis that Transcription Factor 21 is a regulator of steroidogenic factor 1 and is a tumor suppressor gene in pediatric and adult adrenocortical tumors.

New evidences on the regulation of SF-1 expression by POD1/TCF21 in adrenocortical tumor cells

OBJECTIVES: Transcription Factor 21 represses steroidogenic factor 1, a nuclear receptor required for gonadal development, sex determination and the regulation of adrenogonadal steroidogenesis. The aim of this study was to investigate whether silencing or overexpression of the gene Transcription Factor 21 could modulate the gene and protein expression of steroidogenic factor 1 in adrenocortical tumors. METHODS: We analyzed the gene expression of steroidogenic factor 1 using qPCR after silencing endogenous Transcription Factor 21 in pediatric adrenal adenoma-T7 cells through small interfering RNA. In addition, using overexpression of Transcription Factor 21 in human adrenocortical carcinoma cells, we analyzed the protein expression of steroidogenic factor 1 using Western blotting. RESULTS: Transcription Factor 21 knockdown increased the mRNA expression of steroidogenic factor 1 by 5.97-fold in pediatric adrenal adenoma-T7 cells. Additionally, Transcription Factor 21 overexpression inhibited the protein expression of steroidogenic factor 1 by 0.41-fold and 0.64-fold in two different adult adrenocortical carcinoma cell cultures, H295R and T36, respectively. CONCLUSIONS: Transcription Factor 21 is downregulated in adrenocortical carcinoma cells. Taken together, these findings support the hypothesis that Transcription Factor 21 is a regulator of steroidogenic factor 1 and is a tumor suppressor gene in pediatric and adult adrenocortical tumors.

POD-1/TCF21 Reduces SHP Expression, Affecting LRH-1 Regulation and Cell Cycle Balance in Adrenocortical and Hepatocarcinoma Tumor Cells.

POD-1/TCF21 may play a crucial role in adrenal and gonadal homeostasis and represses Sf-1/SF-1 expression in adrenocortical tumor cells. SF-1 and LRH-1 are members of the Fzt-F1 subfamily of nuclear receptors. LRH-1 is involved in several biological processes, and both LRH-1 and its repressor SHP are involved in many types of cancer. In order to assess whether POD-1 can regulate LRH-1 via the same mechanism that regulates SF-1, we analyzed the endogenous mRNA levels of POD-1, SHP, and LRH-1 in hepatocarcinoma and adrenocortical tumor cells using qRT-PCR. Hereafter, these tumor cells were transiently transfected with pCMVMycPod-1, and the effect of POD-1 overexpression on E-box elements in the LRH-1 and SHP promoter region were analyzed by ChIP assay. Also, Cyclin E1 protein expression was analyzed to detect cell cycle progression. We found that POD-1 overexpression significantly decreased SHP/SHP mRNA and protein levels through POD-1 binding to the E-box sequence in the SHP promoter. Decreased SHP expression affected LRH-1 regulation and increased Cyclin E1. These findings show that POD-1/TCF21 regulates SF-1 and LRH-1 by distinct mechanisms, contributing to the understanding of POD-1 involvement and its mechanisms of action in adrenal and liver tumorigenesis, which could lead to the discovery of relevant biomarkers.

POD-1 binding to the E-box sequence inhibits SF-1 and StAR expression in human adrenocortical tumor cells.

Pod-1/Tcf21 is expressed at epithelial-mesenchymal interaction sites during development of many organs. Different approaches have demonstrated that Pod-1 transcriptionally inhibits Sf-1/NR5A1 during gonadal development. Disruption of Sf-1 can lead to disorders of adrenal development, while increased dosage of SF-1 has been related to increased adrenal cell proliferation and tumorigenesis. In this study, we analyzed whether POD-1 overexpression inhibits the endogenous Sf-1 expression in human and mouse adrenocortical tumor cells. Cells were transiently transfected with luciferase reporter gene under the control of Sf-1 promoter and with an expression vector encoding Pod-1. Pod-1 construct inhibited the transcription of the Sf1/Luc reporter gene in a dose-dependent manner in mouse Y-1 adrenocortical carcinoma (ACC) cells, and inhibited endogenous SF-1 expression in the human H295R and ACC-T36 adrenocortical carcinoma cells. These results were validated by chromatin immunoprecipitation assay with POD-1-transfected H295R cells using primers specific to E-box sequence in SF-1 promoter region, indicating that POD-1 binds to the SF-1 E-box promoter. Moreover, POD-1 over-expression resulted in a decrease in expression of the SF-1 target gene, StAR (Steroidogenic Acute Regulatory Protein). Lastly, while the induced expression of POD-1 did not affect the cell viability of H295R/POD-1 or ACC-T36/POD-1 cells, the most significantly enriched KEGG pathways for genes negatively correlated to POD-1/TCF21 in 33 human ACCs were those associated with cell cycle genes.