Embryonic ß-Catenin Is Required for Priming of the Uterus to Implantation.

Repeated implantation failure is a major cause of infertility among healthy women. Uterine ß-catenin (CTNNB1) plays a critical role in implantation. However, the role of embryonic CTNNB1 during implantation remains unclear. We addressed this topic by analyzing mice carrying Ctnnb1-deficient (Ctnnb1Δ/Δ) embryos. Ctnnb1Δ/Δ embryos were produced by intercrossing mice bearing Ctnnb1-deficient eggs and sperms. We found that Ctnnb1Δ/Δ embryos developed to the blastocyst stage; thereafter, they were resorbed, leaving empty decidual capsules. Moreover, leukemia inhibitory factor, a uterine factor essential for implantation, was undetectable in Ctnnb1Δ/Δ blastocysts. Furthermore, CDX2, a transcription factor that determines the fate of trophectoderm cells, was not observed in Ctnnb1Δ/Δ blastocysts. Intrauterine injection with uterine fluids (from control mice) and recombinant mouse leukemia inhibitory factor proteins rescued the uterine response to Ctnnb1Δ/Δ blastocysts. These results suggest that embryonic CTNNB1 is required for the secretion of blastocyst-derived factor(s) that open the implantation window, indicating that the uterine response to implantation can be induced using supplemental materials. Therefore, our results may contribute to the discovery of a similar mechanism in humans, leading to a better understanding of the pathogenesis of repeated implantation failure.

Overdue Calcium Oscillation Causes Polyspermy but Possibly Permits Normal Development in Mouse Eggs.

In some non-mammalian eggs, the fusion of one egg and multiple sperm (polyspermy) induces a robust rise in intracellular calcium ion (Ca2+) concentration due to a shortage of inducers carried by a single sperm. Instead, one of the sperm nuclei is selected inside the egg for normal embryogenesis. Polyspermy also occurs during the in vitro fertilization of human eggs; however, the fate of such eggs is still under debate. Hence, the relationship between polyspermy and repetitive Ca2+ increases (Ca2+ oscillation) in mammals remains unknown. To address this issue, we used mouse sperm lacking extramitochondrial citrate synthase (eCS), which functions as a Ca2+ oscillation inducer; its lack causes retarded Ca2+ oscillation initiation (eCs-KO sperm). Elevated sperm concentrations normalize Ca2+ oscillation initiation. As expected, eCS deficiency enhanced polyspermy in both zona pellucida (ZP)-free and ZP-intact eggs despite producing the next generation of eCs-KO males. In conclusion, similarly to non-mammalian eggs, mouse eggs may develop normally under polyspermy conditions caused by problematic Ca2+ oscillation.

Genetic variants of G-protein coupled receptors associated with pubertal disorders.

Background: The human hypothalamic-pituitary-gonadal (HPG) axis is the regulatory center for pubertal development. This axis involves six G-protein coupled receptors (GPCRs) encoded by KISS1R, TACR3, PROKR2, GNRHR, LHCGR, and FSHR. Methods: Previous studies have identified several rare variants of the six GPCR genes in patients with pubertal disorders. In vitro assays and animal studies have provided information on the function of wild-type and variant GPCRs. Main Findings: Of the six GPCRs, those encoded by KISS1R and TACR3 are likely to reside at the top of the HPG axis. Several loss-of-function variants in the six genes were shown to cause late/absent puberty. In particular, variants in KISS1R, TACR3, PROKR2, and GNRHR lead to hypogonadotropic hypogonadism in autosomal dominant, recessive, and oligogenic manners. Furthermore, a few gain-of-function variants of KISS1R, PROKR2, and LHCGR have been implicated in precocious puberty. The human HPG axis may contain additional GPCRs. Conclusion: The six GPCRs in the HPG axis govern pubertal development through fine-tuning of hormone secretion. Rare sequence variants in these genes jointly account for a certain percentage of genetic causes of pubertal disorders. Still, much remains to be clarified about the molecular network involving the six GPCRs.

Optical Genome Mapping for a Patient with a Congenital Disorder and Chromosomal Translocation.

We performed optical genome mapping (OGM), a newly developed cytogenetic technique, for a patient with a disorder of sex development (DSD) and a 46,XX,t(9;11)(p22;p13) karyotype. The results of OGM were validated using other methods. OGM detected a 9;11 reciprocal translocation and successfully mapped its breakpoints to small regions of 0.9-12.3 kb. OGM identified 46 additional small structural variants, only three of which were detected by array-based comparative genomic hybridization. OGM suggested the presence of complex rearrangements on chromosome 10; however, these variants appeared to be artifacts. The 9;11 translocation was unlikely to be associated with DSD, while the pathogenicity of the other structural variants remained unknown. These results indicate that OGM is a powerful tool for detecting and characterizing chromosomal structural variations, although the current methods of OGM data analyses need to be improved.

Mosaic loss of the Y chromosome and men's health.

Background: Although Y chromosomal genes are involved in male sex development, spermatogenesis, and height growth, these genes play no role in the survival or mitosis of somatic cells. Therefore, somatic cells lacking the Y chromosome can stay and proliferate in the body. Methods: Several molecular technologies, including next-generation sequencing and multiplex PCR-based assays, are used to detect mosaic loss of the Y chromosome (mLOY) in the blood of men. Main findings: Accumulating evidence suggests that mLOY represents the most common acquired chromosomal alteration in humans, affecting >40% of men over 70 years of age. Advanced age, tobacco smoking, and some SNPs in cell cycle genes are known to increase the frequency of mLOY. The developmental process of mLOY in elderly men remains to be clarified, but it possibly reflects recurrent mitotic elimination of Y chromosomes or clonal expansion of 45,X cell lineages. In rare cases, mLOY also occurs in young men and fetuses. MLOY has been associated with early death, cancers, and other disorders in elderly men, infertility in reproductive-aged men, and developmental defects in children. Conclusion: Y chromosomes in men can be lost at every life stage and Y chromosomal loss is associated with various health problems.

Structural and numerical Y chromosomal variations in elderly men identified through multiplex ligation-dependent probe amplification.

Human Y chromosomes frequently acquire structural and numerical alterations. Known alterations include germline copy-number variations (CNVs) in the azoospermia factor (AZF) region and somatic mosaic loss of the Y chromosome (mLOY). Here, we explored Y chromosomal variations in 160 Japanese men aged 75-90 years. Multiplex ligation-dependent probe amplification (MLPA) identified ten types of AZF-linked CNVs in 77 men and mLOY of various degrees in 37. Seventeen men carried both a CNV and mLOY. MLOY levels estimated by MLPA were closely correlated with those determined by droplet digital PCR. No association was found between AZF-linked CNVs and the frequency or levels of mLOY. These results emphasize the high frequency and large inter-individual variability of AZF-linked CNVs and mLOY, and demonstrate the usefulness of MLPA in the detection of these variations. More importantly, this study provides the first evidence that AZF-linked CNVs do not increase the risk of aging-related mLOY.

Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.

Men and women become infertile with age, but the mechanism of declining male fertility, more specifically, the decrease in in sperm quality, is not well known. Citrate synthase (CS) is a core enzyme of the mitochondrial tricarboxylic acid (TCA) cycle, which directly controls cellular function. Extra-mitochondrial CS (eCS) is produced and abundant in the sperm head; however, its role in male fertility is unknown. We investigated the role of eCS in male fertility by producing eCs-deficient (eCs-KO) mice. The initiation of the first spike of Ca2+ oscillation was substantially delayed in egg fused with eCs-KO sperm, despite normal expression of sperm factor phospholipase C zeta 1. The eCs-KO male mice were initially fertile, but the fertility dropped with age. Metabolomic analysis of aged sperm revealed that the loss of eCS enhances TCA cycle in the mitochondria with age, presumably leading to depletion of extra-mitochondrial citrate. The data suggest that eCS suppresses age-dependent male infertility, providing insights into the decline of male fertility with age.

Correction: Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Germline-Derived Gain-of-Function Variants of Gsα-Coding GNAS Gene Identified in Nephrogenic Syndrome of Inappropriate Antidiuresis.

BACKGROUND: The stimulatory G-protein α-subunit encoded by GNAS exons 1-13 (GNAS-Gsα) mediates signal transduction of multiple G protein-coupled receptors, including arginine vasopressin receptor 2 (AVPR2). Various germline-derived loss-of-function GNAS-Gsα variants of maternal and paternal origin have been found in pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism, respectively. Specific somatic gain-of-function GNAS-Gsα variants have been detected in McCune-Albright syndrome and may result in phosphate wasting. However, no germline-derived gain-of-function variant has been identified, implying that such a variant causes embryonic lethality. METHODS: We performed whole-exome sequencing in two families with dominantly inherited nephrogenic syndrome of inappropriate antidiuresis (NSIAD) as a salient phenotype after excluding a gain-of-function variant of AVPR2 and functional studies for identified variants. RESULTS: Whole-exome sequencing revealed two GNAS-Gsα candidate variants for NSIAD: GNAS-Gsα p.(F68_G70del) in one family and GNAS-Gsα p.(M255V) in one family. Both variants were absent from public and in-house databases. Of genes with rare variants, GNAS-Gsα alone was involved in AVPR2 signaling and shared by the families. Protein structural analyses revealed a gain-of-function-compatible conformational property for p.M255V-Gsα, although such assessment was not possible for p.F68_G70del-Gsα. Both variants had gain-of-function effects that were significantly milder than those of McCune-Albright syndrome-specific somatic Gsα variants. Model mice for p.F68_G70del-Gsα showed normal survivability and NSIAD-compatible phenotype, whereas those for p.M255V-Gsα exhibited severe failure to thrive. CONCLUSIONS: This study shows that germline-derived gain-of-function rare variants of GNAS-Gsα exist and cause NSIAD as a novel Gsα-mediated genetic disease. It is likely that AVPR2 signaling is most sensitive to GNAS-Gsα's gain-of-function effects.

Suppression of Non-Random Fertilization by MHC Class I Antigens.

Hermaphroditic invertebrates and plants have a self-recognition system on the cell surface of sperm and eggs, which prevents their self-fusion and enhances non-self-fusion, thereby contributing to genetic variation. However, the system of sperm-egg recognition in mammals is under debate. To address this issue, we explored the role of major histocompatibility complex class I (MHC class I, also known as histocompatibility 2-Kb or H2-Kb and H2-Db in mice) antigens by analyzing H2-Kb-/-H2-Db-/-ß2-microglobulin (ß2M)-/- triple-knockout (T-KO) male mice with full fertility. T-KO sperm exhibited an increased sperm number in the perivitelline space of wild-type (WT) eggs in vitro. Moreover, T-KO sperm showed multiple fusion with zona pellucida (ZP)-free WT eggs, implying that the ability of polyspermy block for sperm from T-KO males was weakened in WT eggs. When T-KO male mice were intercrossed with WT female mice, the percentage of females in progeny increased. We speculate that WT eggs prefer fusion with T-KO sperm, more specifically X-chromosome-bearing sperm (X sperm), suggesting the presence of preferential (non-random) fertilization in mammals, including humans.

Copy-number analysis of Y-linked loci in young men with non-obstructive azoospermia: Implications for the rarity of early onset mosaic loss of chromosome Y.

PURPOSE: Mosaic loss of chromosome Y (mLOY) is a common feature in elderly men. If mLOY can also occur in young men, it may lead to spermatogenic failure due to loss of spermatogenic genes. Indeed, previous studies detected the 45,X/46,XY karyotype in a few young men with spermatogenic failure. The present study aimed to clarify the frequency of cryptic mLOY in reproductive-aged men with spermatogenic failure. METHODS: We studied 198 men at ages 24-55 years who presented with etiology-unknown non-obstructive azoospermia. Prior this study, these patients underwent G-banding analysis for 20 leukocytes and were found to have 46,XY karyotype. We analyzed copy numbers of chromosome Y in blood cells by using semi-quantitative multiplex PCR for AMELY/AMELX, array-based comparative genomic hybridization (CGH) for the AMELY locus, and droplet digital PCR for SRY, USP9Y, and UTY. RESULTS: Multiplex PCR showed borderline low AMELY/AMELX ratios in three patients. However, for the three patients, CGH excluded deletion of the AMELY locus, and droplet digital PCR suggested preserved copy numbers of all tested loci. CONCLUSION: This study highlights the rarity of leukocyte mLOY in reproductive-aged men with spermatogenic failure. In addition, our data imply that standard karyotyping is sufficient to screen early onset mLOY.

Membrane protein CD9 is repositioned and released to enhance uterine function.

Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.

DNA Methylation Status of SHOX-Flanking CpG Islands in Healthy Individuals and Short Stature Patients with Pseudoautosomal Copy Number Variations.

SHOX resides in the short arm pseudoautosomal region (PAR1) of the sex chromosomes and escapes X inactivation. SHOX haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). A substantial percentage of cases with SHOX haploinsufficiency arise from pseudoautosomal copy number variations (CNVs) involving putative enhancer regions of SHOX. Our previous study using peripheral blood samples showed that some CpG dinucleotides adjacent to SHOX exon 1 were hypomethylated in a healthy woman and methylated in a woman with gross X chromosomal rearrangements. However, it remains unknown whether submicroscopic pseudoautosomal CNVs cause aberrant DNA methylation of SHOX-flanking CpG islands. In this study, we examined the DNA methylation status of SHOX-flanking CpG islands in 50 healthy individuals and 10 ISS/LWD patients with pseudoautosomal CNVs. In silico analysis detected 3 CpG islands within the 20-kb region from the translation start site of SHOX. Pyrosequencing and bisulfite sequencing of genomic DNA samples revealed that these CpG islands were barely methylated in peripheral blood cells and cultured chondrocytes of healthy individuals, as well as in peripheral blood cells of ISS/LWD patients with pseudoautosomal CNVs. These results, in conjunction with our previous findings, indicate that the DNA methylation status of SHOX-flanking CpG islands can be affected by gross X-chromosomal abnormalities, but not by submicroscopic CNVs in PAR1. Such CNVs likely disturb SHOX expression through DNA methylation-independent mechanisms, which need to be determined in future studies.

Degradation of phosphate polymer polyP enhances lactic fermentation in mice.

In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a "molecular fossil" due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

Endometrial preparation methods for frozen-thawed embryo transfer are associated with altered risks of hypertensive disorders of pregnancy, placenta accreta, and gestational diabetes mellitus.

STUDY QUESTION: What were the risks with regard to the pregnancy outcomes of patients who conceived by frozen-thawed embryo transfer (FET) during a hormone replacement cycle (HRC-FET)? SUMMARY ANSWER: The patients who conceived by HRC-FET had increased risks of hypertensive disorders of pregnancy (HDP) and placenta accreta and a reduced risk of gestational diabetes mellitus (GDM) in comparison to those who conceived by FET during a natural ovulatory cycle (NC-FET). WHAT IS KNOWN ALREADY: Previous studies have shown that pregnancy and live-birth rates after HRC-FET and NC-FET are comparable. Little has been clarified regarding the association between endometrium preparation and other pregnancy outcomes. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of patients who conceived after HRC-FET and those who conceived after NC-FET was performed based on the Japanese assisted reproductive technology registry in 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: The pregnancy outcomes were compared between NC-FET (n = 29 760) and HRC-FET (n = 75 474) cycles. Multiple logistic regression analyses were performed to investigate the potential confounding factors. MAIN RESULTS AND THE ROLE OF CHANCE: The pregnancy rate (32.1% vs 36.1%) and the live birth rate among pregnancies (67.1% vs 71.9%) in HRC-FET cycles were significantly lower than those in NC-FET cycles. A multiple logistic regression analysis showed that pregnancies after HRC-FET had increased odds of HDPs [adjusted odds ratio, 1.43; 95% confidence interval (CI), 1.14-1.80] and placenta accreta (adjusted odds ratio, 6.91; 95% CI, 2.87-16.66) and decreased odds for GDM (adjusted odds ratio, 0.52; 95% CI, 0.40-0.68) in comparison to pregnancies after NC-FET. LIMITATIONS, REASONS FOR CAUTION: Our study was retrospective in nature, and some cases were excluded due to missing data. The implication of bias and residual confounding factors such as body mass index, alcohol consumption, and smoking habits should be considered in other observational studies. WIDER IMPLICATIONS OF THE FINDINGS: Pregnancies following HRC-FET are associated with higher risks of HDPs and placenta accreta and a lower risk of GDM. The association between the endometrium preparation method and obstetrical complication merits further attention. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained for this work. The authors declare no conflicts of interest in association with the present study. TRIAL REGISTRATION NUMBER: Not applicable.

SHOX far-downstream copy-number variations involving cis-regulatory nucleotide variants in two sisters with Leri-Weill dyschondrosteosis.

SHOX haploinsufficiency leading to Leri-Weill dyschondrosteosis (LWD) and idiopathic short stature typically results from intragenic mutations or copy-number variations (CNVs) involving SHOX and/or its putative enhancer regions that are distributed in the genomic interval between 400 kb and 840 kb from Xpter/Ypter. Here, we report two sisters with LWD, who carried a deletion in the far-downstream region of SHOX. The 0.62 Mb deletion contained 50 single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels), whose genotypes were linked to SHOX expression levels in the Genotype-Tissue Expression portal. Notably, most of these SNPs/indels accumulated within a ~20 kb interval that was positioned ~900 kb away from Xpter/Ypter. These SNPs/indels showed similar minor allele frequencies, indicating that they reside within a haplotype block. The ~20 kb interval was not evolutionarily conserved; however, it was associated with the previously determined peak of chromosome conformation capture profiling (4C)-seq. Importantly, the deletion in the present cases partially overlapped with CNVs of three previous cases with skeletal deformity and/or short stature. The results indicate that far-downstream CNVs constitute rare genetic causes of SHOX haploinsufficiency. These CNVs possibly impair SHOX expression through copy-number changes of a human-specific cis-regulatory haplotype block. This notion awaits further validation.

Dihydrotestosterone induces minor transcriptional alterations in genital skin fibroblasts of children with and without androgen insensitivity.

Endogenous and exogenous androgens induce masculinization of external genitalia through binding to the androgen receptor (AR). The target genes of androgens in external genitalia remain to be determined, although previous studies have shown that the apolipoprotein D gene (APOD) was significantly upregulated by dihydrotestosterone (DHT), the most potent androgen in humans. In the present study, we performed microarray analysis for genital skin fibroblasts obtained from four boys with buried penis (the control individuals) and a patient with partial androgen insensitivity syndrome (PAIS) due to a hypomorphic mutation in AR (the PAIS patient). We identified 24 transcripts that were upregulated or downregulated by DHT in all samples of control individuals and, to a lesser extent, in the sample of the PAIS patient. Differences between DHT-treated and -untreated samples were small; the results of 24 transcripts did not reach statistical significance. The 24 transcripts included CYP1B1, a gene possibly involved in the development of genital tubercle in mice, and APOD, as well as several genes that have been reported as androgen targets in prostate or other tissues. The results of this study indicate that androgen-mediated masculinization of external genitalia is unlikely to depend on massive transcriptional changes in specific AR target genes. Rather, minor transcriptional changes of several genes, and/or a complex molecular network may play a major role in penile development. Importantly, our data suggest the possible involvement of CYP1B1 in human genital development and confirm the clinical importance of APOD as a biomarker for AR function.

STX2 is a causative gene for nonobstructive azoospermia.

STX2 encodes a sulfoglycolipid transporter. Although Stx2 nullizygosity is known to cause spermatogenic failure in mice, STX2 mutations have not been identified in humans. Here, we performed STX2 mutation analysis for 131 Japanese men clinically diagnosed with nonobstructive azoospermia. As a result, we identified a homozygous frameshift mutation [c.8_12delACCGG, p.(Asp3Alafs*8)] in one patient. The mutation-positive patient exhibited loss-of-heterozygosity for 58.4 Mb genomic regions involving STX2, suggesting possible parental consanguinity. The patient showed azoospermia, relatively small testes, and a mildly elevated follicle stimulating hormone level, but no additional clinical features. Testicular histology of the patient showed universal maturation arrest and multinucleated spermatocytes, which have also been observed in mice lacking Stx2. PCR-based cDNA screening revealed wildtype STX2 expression in various tissues including the testis. Our results indicate that STX2 nullizygosity results in nonsyndromic maturation arrest with multinucleated spermatocytes, and accounts for a small fraction of cases with nonobstructive azoospermia.

Somatically Acquired Isodicentric Y and Mosaic Loss of Chromosome Y in a Boy with Hypospadias.

Isodicentric Y chromosome [idic(Y)] represents a relatively common subtype of Y chromosomal rearrangements in the germline; however, limited evidence supports the postzygotic occurrence of idic(Y). Here, we report a boy with hypospadias and somatically acquired idic(Y). The 3.5-year-old boy has been identified in our previous study for patients with hypospadias. In the present study, cytogenetic analysis including FISH revealed a 45,X[5]/46,X,idic(Y)[7]/46,XY[8] karyotype. MLPA showed a mosaic deletion involving PPP1R12BP1 and RBMY2DP. The idic(Y) was likely to have been formed through aberrant recombination between P1 palindromes and subsequently underwent mosaic loss. The patient's phenotype was attributable to deletion of some Y chromosomal genes and/or mosaic loss of chromosome Y (mLOY). The results suggest that idic(Y) can originate in postzygotic cells via palindrome-mediated crossovers. Moreover, our data indicate that somatically acquired idic(Y) can trigger mLOY, which usually appears as an aging-related phenomenon in elderly men.

Gain-of-function mutations in G-protein-coupled receptor genes associated with human endocrine disorders.

The human genome encodes more than 700 G-protein-coupled receptors (GPCRs), many of which are involved in hormone secretion. To date, more than 100 gain-of-function (activating) mutations in at least ten genes for GPCRs, in addition to several loss-of-function mutations, have been implicated in human endocrine disorders. Previously reported gain-of-function GPCR mutations comprise various missense substitutions, frameshift mutations, intragenic inframe deletions and copy-number gains. Such mutations appear in both germline and somatic tumour cells, and lead to various hormonal abnormalities reflecting excessive receptor activity. Phenotypic consequences of these mutations include distinctive endocrine syndromes, as well as relatively common hormonal abnormalities. Such mutations encode hyperfunctioning receptors with increased constitutive activity, broadened ligand specificity, increased ligand sensitivity and/or delayed receptor desensitization. Furthermore, recent studies proposed a paradoxical gain-of-function mechanism caused by inactive GPCR mutants. Molecular diagnosis of GPCR activating mutations serves to improve the clinical management of mutation-positive patients. This review aims to introduce new aspects regarding gain-of-function mutations in GPCR genes associated with endocrine disorders.