Abnormal hypothalamic/posterior pituitary development appears to be a major determinant of pituitary stalk interruption syndrome (PSIS). The observation of familial cases and associated congenital ...abnormalities suggests a genetic basis. Single‐gene mutations explain less than 5% of the cases, and whole exome sequencing has shown heterogeneous results. The present study aimed to assess copy number variation (CNV) using array‐based comparative genomic hybridization (aCGH) in patients with non‐syndromic PSIS and comprehensively review data from the literature on CNV analysis in congenital hypopituitarism (CH) patients. Twenty‐one patients with sporadic CH from our outpatient clinics presented with ectopic posterior pituitary (EPP) and no central nervous system abnormalities on magnetic resonance image (MRI) or any other malformations on physical examination at presentation were enrolled in the study. aCGH using a whole‐genome customized 400K oligonucleotide platform was performed in our patients. For the literature review, we searched for case reports of patients with CH and CNV detected by either karyotype or aCGH reported in PubMed up to November 2021. Thirty‐five distinct rare CNVs were observed in 18 patients (86%) and two of them (6%) were classified as pathogenic: one deletion of 1.8 Mb in chromosome 17 (17q12) and one deletion of 15 Mb in chromosome 18 (18p11.32p11.21), each one in a distinct patient. In the literature review, 67 pathogenic CNVs were published in 83 patients with CH, including the present study. Most of these patients had EPP (78% out of the 45 evaluated by sellar MRI) and were syndromic (70%). The most frequently affected chromosomes were X, 18, 20 and 1. Our study has found that CNV can be a mechanism of genetic abnormality in non‐syndromic patients with CH and EPP. In future studies, one or more genes in those CNVs, both pathogenic and variant of uncertain significance, may be considered as good candidate genes.
Pituitary development takes place from two embryonal apposed ectodermal tissues. The anterior pituitary (AP), so‐called adenohypophysis, forms from an upward protrusion of the epithelium of the roof of the mouth (Rathke's pouch, Rp) that extends toward the posterior pituitary (PP), or neurohypophysis, which grows downward by a ventral evagination from the diencephalon (de) of the developing brain. PP is attached to the infundibular (inf) stalk, which arises from an anlage in the floor of the third ventricle. Ectopic posterior pituitary (EPP) is the hallmark of pituitary stalk interruption syndrome (PSIS), which is mainly associated with AP endocrine defects because of its early neural induction of the Rp. However, pituitary‐development single‐gene mutations explain less than 5% of the cases, and whole exome sequencing still presents heterogeneous results. Our findings demonstrate the contribution of searching for copy number variation (CNV) using array‐based comparative genomic hybridization (aCGH) in patients with PSIS, suggesting that CNV can be an additional mechanism of developmental abnormality in non‐syndromic patients with congenital hypopituitarism and ectopic posterior pituitary.
Thyrotoxic hypokalemic periodic paralysis (TPP) is characterized by acute attacks of weakness, hypokalemia, and thyrotoxicosis of various etiologies. These transient attacks resemble those of ...patients with familial hypokalemic periodic paralysis (hypoKPP) and resolve with treatment of the underlying hyperthyroidism. Because of the phenotypic similarity of these conditions, we hypothesized that TPP might also be a channelopathy. While sequencing candidate genes, we identified a previously unreported gene (not present in human sequence databases) that encodes an inwardly rectifying potassium (Kir) channel, Kir2.6. This channel, nearly identical to Kir2.2, is expressed in skeletal muscle and is transcriptionally regulated by thyroid hormone. Expression of Kir2.6 in mammalian cells revealed normal Kir currents in whole-cell and single-channel recordings. Kir2.6 mutations were present in up to 33% of the unrelated TPP patients in our collection. Some of these mutations clearly alter a variety of Kir2.6 properties, all altering muscle membrane excitability leading to paralysis.
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KCNJ18 mutations are associated with thyrotoxic hypokalemic periodic paralysis, TPP ►
KCNJ18 encodes an inward rectifying potassium channel, Kir 2.6 ► Thyroid hormones regulate KCNJ18 both transcriptionally and posttranscriptionally
Genetic testing for germline RET pathogenic variants, which cause the Multiple Endocrine Neoplasia Type 2 (MEN2) syndrome, has become crucial in managing patients with medullary thyroid carcinoma ...(MTC). Classically, RET heterozygous missense pathogenic variants are transmitted in a Mendelian autosomal dominant pattern, of which germline/gonadal mosaicism has never been reported. We report the novel occurrence of a MEN2A patient's family in which the siblings inherited three different RET 634 genotypes: wild type (p.Cys634), p.Cys634Gly or p.Cys634Arg heterozygous pathogenic variants. We hypothesized that germline/gonadal mosaicism, derived from an inherited + early somatic mutation in the mother or a double de novo mutation during maternal embryogenesis, led to this rare event in the RET gene. Exome analysis of the proband's deceased mother's paraffin‐embedded thyroid tissue confirmed the three nucleotides in the same 634 codon position. For the first time, we describe germline/gonadal mosaicism in RET, generating a second pathogenic amino acid change in the same codon causing MEN2A. Our finding shows that RET parental mosaicism, confirmed by somatic exome sequencing, might explain discrepant genotype cases in siblings with inherited cancers.
Abstract Reproductive physiology involves complex biological processes that can be disrupted by exposure to environmental contaminants. The effects of bisphenol A (BPA) on spermatogenesis and sperm ...quality is still unclear. The objective of this study was to investigate the reproductive toxicity of BPA at dosages considered to be safe (5 or 25 mg BPA/kg/day). We assessed multiple sperm parameters, the relative expression of genes involved in the central regulation of the hypothalamic–pituitary–testicular axis, and the serum concentrations of testosterone, estradiol, LH and FSH. BPA exposure reduced sperm production, reserves and transit time. Significant damage to the acrosomes and the plasma membrane with reduced mitochondrial activity and increased levels of defective spermatozoa may have compromised sperm function and caused faster movement through the epididymis. BPA exposure reduced the serum concentrations of testosterone, LH and FSH and increased the concentration of estradiol. The relative gene expression revealed an increase in gonadotropin releasing hormone receptor ( Gnrhr ), luteinizing hormone beta ( Lhb ), follicle stimulating hormone beta ( Fshb ), estrogen receptor beta ( Esr2 ) and androgen receptor ( Ar ) transcripts in the pituitary and a reduction in estrogen receptor alpha ( Esr1 ) transcripts in the hypothalamus. In this study, we demonstrated for the first time that adult male exposure to BPA caused a reduction in sperm production and specific functional parameters. The corresponding pattern of gene expression is indicative of an attempt by the pituitary to reestablish normal levels of LH, FSH and testosterone serum concentrations. In conclusion, these data suggest that at dosages previously considered nontoxic to reproductive function, BPA compromises the spermatozoa and disrupts the hypothalamic–pituitary–gonadal axis, causing a state of hypogonadotropic hypogonadism.
Congenital hypogonadotrophic hypogonadism (CHH) is a challenging inherited endocrine disorder characterised by absent or incomplete pubertal development and infertility as a result of the low ...action/secretion of the hypothalamic gonadotrophin‐releasing hormone (GnRH). Given a growing list of gene mutations accounting for CHH, the application of massively parallel sequencing comprises an excellent molecular diagnostic approach because it enables the simultaneous evaluation of many genes. The present study proposes the use of whole exome sequencing (WES) to identify causative and modifying mutations based on a phenotype‐genotype CHH analysis using an in‐house exome pipeline. Based on 44 known genes related to CHH in humans, we were able to identify a novel homozygous gonadotrophin‐releasing hormone receptor (GNRHR) p.Thr269Met mutant, which segregates with the CHH kindred and was predicted to be deleterious by in silico analysis. A functional study measuring intracellular inositol phosphate (IP) when stimulated with GnRH on COS‐7 cells confirmed that the p.Thr269Met GnRHR mutant performed greatly diminished IP accumulation relative to the transfected wild‐type GnRHR. Additionally, the proband carries three heterozygous variants in CCDC141 and one homozygous in SEMA3A gene, although their effects with respect to modifying the phenotype are uncertain. Because they do not segregate with reproductive phenotype in family members, we advocate they do not contribute to CHH oligogenicity. WES proved to be useful for CHH molecular diagnosis and reinforced its benefit with respect to identifying heterogeneous genetic disorders. Our findings expand the GnRHR mutation spectrum and phenotype‐genotype correlation in CHH.
Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) that is widely used in the manufacturing of plastics and inner linings of food cans. Previously, it was reported that BPA disturbed the ...sexual dimorphic nucleus of the hypothalamus and delaying the onset of puberty attributed to an estrogenic action. In addition, BPA during the perinatal period increased LH serum concentrations in male offspring of dams at doses below the reproductive NOAEL (No Observable Adverse Effect Level) based upon World Health Organization guidelines. Based upon these findings, the objective of this study was to (1) determine the effects of perinatal treatment with low doses of BPA on regulation of spermatogenesis in adult offspring and (2) elucidate molecular mechanisms involved in the pathogenesis of gonadal dysfunction. The expression of genes related to spermatogenesis was disrupted with adverse consequences on sperm production, reserves, and function. Both BPA treated groups exhibited reduction in sperm production and epithelial height of seminiferous tubules, accompanied by diminished integrity of the acrosome and plasma membrane, decreased mitochondrial activity and increased incidence of morphological abnormalities. The sperm transit time was also slower. However, only in the group receiving the higher BPA dose was transcript expression of genes affected (reduced Ar and increased Esr1). It is of interest that serum testosterone levels were elevated in the same group where Ar was decreased. Data suggest that exposure to low BPA doses during hypothalamic sexual differentiation period produces permanent deleterious effects on spermatogenesis in adulthood.
Abstract
Context
Thyroid dysgenesis (TD) is the leading cause of congenital hypothyroidism (CH). The etiology of TD remains unknown in ∼90% of cases, the most common form being thyroid ectopia (TE) ...(48% to 61%).
Objective
To search for candidate genes in hypothyroid children with TE.
Design, Setting, and Participants
We followed a cohort of 268 children with TD and performed whole-exome sequencing (WES) in three children with CH with TE (CHTE) and compared them with 18 thyroid-healthy controls. We then screened an additional 41 children with CHTE by Sanger sequencing and correlated the WES and Sanger molecular findings with in vitro functional analysis.
Main Outcome Measures
Genotyping, mutation prediction analysis, and in vitro functional analysis.
Results
We identified seven variants in the DUOX2 gene, namely G201E, L264CfsX57, P609S, M650T, E810X, M822V, and E1017G, and eight known variations. All children carrying DUOX2 variations had high thyroid-stimulating hormone levels at neonatal diagnosis. All mutations were localized in the N-terminal segment, and three of them led to effects on cell surface targeting and reactive oxygen species generation. The DUOX2 mutants also altered the interaction with the maturation factor DUOXA2 and the formation of a stable DUOX2/DUOXA2 complex at the cell surface, thereby impairing functional enzymatic activity. We observed no mutations in the classic genes related to TD or in the DUOX1 gene.
Conclusion
Our findings suggest that, in addition to thyroid hormonogenesis, the DUOX2 N-terminal domain may play a role in thyroid development.
Exome analysis unexpectedly revealed DUOX2 mutations in patients with ectopic thyroid, suggesting that DUOX2 could also be involved in thyroid ontogenesis.
X-chromosome inactivation occurs randomly in normal female cells. However, the inactivation can be skewed in patients with alterations in X-chromosome. In balanced X-autosome translocations, normal X ...is preferentially inactivated, while in unbalanced X alterations, the aberrant X is usually inactivated. Here, we present a novel strategy to verify the skewed X inactivation pattern through the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into cells, in 11 patients: five carriers of balanced X-autosome translocations and six of unbalanced X-chromosome alterations. Since EdU is a labeled nucleoside analog of thymidine, its incorporation during DNA synthesis can reveal late replication regions and the inactive X-chromosome. All EdU findings were validated by the human androgen receptor gene (HUMARA) assay. The late replication regions were easily and quickly visualized in all cells, where inactive Xs are marked with strong green fluorescence. It was observed that the normal X-chromosome was preferentially inactivated in patients with balanced X-autosome translocations; while the aberrant X-chromosome was inactivated in most cells from patients with unbalanced alterations. By performing the fluorescence-based EdU assay, the differences between the active and inactive X-chromosomes are more easily recognizable than by classic cytogenetic methods. Furthermore, EdU incorporation allows the observation of the late replication regions in autosomal segments present in X derivatives from X-autosome translocations. Therefore, EdU assay permits an accurate and efficient cytogenetic evaluation of the X inactivation pattern with a low-cost, easy to perform and highly reproducible technique.
The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants ...in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored.
By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells.
Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.
Summary
Objective
Staging systems applied to medullary thyroid cancer (MTC) rely on initial clinical and pathological features and do not consider the response to treatment. To determine whether MTC ...staging can be improved by incorporating the first postoperative calcitonin measurement.
Patients and measurements
Eighty‐five patients being monitored for MTC (median follow‐up 5 years) were retrospectively classified according to both the American Joint Committee on Cancer (AJCC) and the proposed combined risk stratification system (low, intermediate and high risk), which incorporates the first postoperative calcitonin measurement, using the outcomes no evidence of disease (NED), biochemical evidence of disease, structurally identifiable disease and death.
Results
Ninety per cent of AJCC I patients were classified as NED at final follow‐up. When we added a postoperative calcitonin measurement, 95% low‐risk patients were classified as NED at final follow‐up. AJCC stages I and IV were associated, respectively, with no occurrence and a high rate (63%) of structurally identifiable disease. Stages II and III yielded similar predictions of structurally identifiable disease, 13% and 14%, respectively. When we included the postoperative calcitonin level, the patients with structural evidence of disease included none from the low‐risk group, 10% from the intermediate group and 63% from the high‐risk group. The proportion of variance explained analysis (PVE) was better for the combined risk stratification system (54%) than for the AJCC system alone (32%).
Conclusion
Including the first postoperative calcitonin measurement with the anatomical staging system can better predict the clinical outcome of patients with MTC and refine the follow‐up of these patients.
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