RET 634 germline/gonadal mosaicism generating a second pathogenic amino acid change in multiple endocrine neoplasia type 2A.

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.

Cosegregation of a novel mutation in the sixth transmembrane segment of the luteinizing/choriogonadotropin hormone receptor with two Brazilian siblings with severe testotoxicosis.

PURPOSE: Testotoxicosis is an autosomal dominant form of gonadotropin-independent precocious puberty caused by heterozygous constitutively activating mutations of the luteinizing hormone/choriogonadotropin receptor (LHCGR) gene. The aim of this study was to describe two Brazilian siblings with testotoxicosis, to confirm the molecular diagnosis, and to perform an in silico analysis of a novel mutation in the hot spot of the LHCGR gene. MATERIALS AND METHODS: Molecular analysis of the mutation on the LHCGR gene was performed by direct Sanger sequencing, followed by an in silico analysis using HOPE bioinformatics tool to predict a functional defect of the mutant. RESULTS: Both patients presented with gonadotropin-independent precocious puberty before the age of four years. Genetic analysis revealed a novel non-maternally inherited p.Asp578Val mutation of the LHCGR gene. An in silico analysis showed that the p.Asp578Val mutation disturbed amino acid physicochemical features regarding its size, charge, and hydrophobicity value. CONCLUSIONS: Clinical and hormonal profile of the siblings here evaluated was not different while compared to those patients previously described. An in silico mutation analysis reinforced the causative role of recurrent activating mutations in the intracellular loop and transmembrane helices of the LHCGR. The segregation of this mutation with the offsprings' phenotype indicated that it is causative.

Whole genome and exome sequencing realignment supports the assignment of KCNJ12, KCNJ17, and KCNJ18 paralogous genes in thyrotoxic periodic paralysis locus: functional characterization of two polymorphic Kir2.6 isoforms.

Next-generation sequencing (NGS) has enriched the understanding of the human genome. However, homologous or repetitive sequences shared among genes frequently produce dubious alignments and can puzzle NGS mutation analysis, especially for paralogous potassium channels. Potassium inward rectifier (Kir) channels are important to establish the resting membrane potential and regulating the muscle excitability. Mutations in Kir channels cause disorders affecting the heart and skeletal muscle, such as arrhythmia and periodic paralysis. Recently, a susceptibility muscle channelopathy-thyrotoxic periodic paralysis (TPP)-has been related to Kir2.6 channel (KCNJ18 gene). Due to their high nucleotide sequence homology, variants found in the potassium channels Kir2.6 and Kir2.5 have been mistakenly attributable to Kir2.2 polymorphisms or mutations. We aimed at elucidating nucleotide misalignments by performing realignment of whole exome sequencing (WES) and whole genome sequencing (WGS) reads to specific Kir2.2, Kir2.5, and Kir2.6 cDNA sequences using BWA-MEM/GATK pipeline. WES/WGS reads correctly aligned 26.9/43.2, 37.6/31.0, and 35.4/25.8 % to Kir2.2, Kir2.5, and Kir2.6, respectively. Realignment was able to reduce over 94 % of misalignments. No putative mutations of Kir2.6 were identified for the three TPP patients included in the cohort of 36 healthy controls using either WES or WGS. We also distinguished sequences for a single Kir2.2, a single Kir2.5 sequence, and two Kir2.6 isoforms, which haplotypes were named RRAI and QHEV, based on changes at 39, 40, 56, and 249 residues. Electrophysiology records on both Kir2.6_RRAI and _QHEV showed typical rectifying currents. In our study, the reduction of misalignments allowed the elucidation of paralogous gene sequences and two distinct Kir2.6 haplotypes, and pointed the need for checking the frequency of these polymorphisms in other populations with different genetic background.

COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Assessing the clinical and molecular diagnosis of inherited forms of impaired sensitivity to thyroid hormone from a single tertiary center.

AIM: Resistance to thyroid hormone (RTH), characterized by persistent hyperthyroxinemia with non-suppressed thyrotropin (TSH), is mostly caused by mutations in thyroid hormone receptor beta gene (THRB). Two differential diagnoses should be considered due to similar clinical and laboratory findings: TSH-producing pituitary adenoma (TPA) and Familial Dysalbuminemic Hyperthyroxinemia (FDH). The aim of this study is to describe our single tertiary center experience in the molecular diagnosis of RTH in Brazilian patients, analyzing their clinical and laboratory characteristics and the most common differential diagnosis. SUBJECTS AND METHODS: We enrolled 30 subjects with clinical and laboratory features of RTH. Patient´s evaluations included clinical examination, thyroid hormone profile and imaging tests. Sequencing analysis for THRB hot spot region was conducted on all patients, and those without mutations in beta isoform of the thyroid hormone receptor (TRß) (non-TR-RTH) were investigated for albumin gene (ALB) mutation. RESULTS: Seventeen patients presented mutations in TRß (RTHß); six were non-TR-RTH, three had a diagnosis of FDH with a mutation in ALB, and four were diagnosed with TPA. Two characteristics were different to what is commonly described in the literature: higher serum TSH levels in RTHß patients when compared to the non-TR-RTH group, but this difference did not extend to free T4 (FT4) level; also the percentage of non-TR-RTH was higher than what was reported in other series. CONCLUSION: In the present series, most cases were RTHß with higher levels of TSH. We described three novel mutations in THRB (p.M313V, p.R320G and p.R438P) and the first patients with FDH molecular diagnosis (p.R242H) documented in Brazil.

Novel lincRNA Susceptibility Gene and Its Role in Etiopathogenesis of Thyrotoxic Periodic Paralysis.

Thyrotoxic periodic paralysis (TPP) is a life-threatening neuromuscular complication of thyrotoxicosis characterized by muscle weakness and hypokalemia and with an unclear etiopathogenesis. However, the 17q24.3 locus had been genetically linked to TPP, in which the genetic variant rs312691 (TC genotype) in long intergenic noncoding RNA (lincRNA) CTD-2378E21.1 is located downstream of inward-rectifier potassium (Kir) channel genes [KCNJ2 and its antisense KCNJ2 (AS-KCNJ2)]. A TPP patient with a suppressed thyroid-stimulating hormone level, a high free thyroxine level of (5.8 ng/dL), and low serum potassium level of (2 mEq/L) was evaluated for Kir channel expression during and after recovery from thyrotoxicosis. We observed that circulating lincRNA and Kir expression varied in accordance with thyroid status and TC genotype. To endorse this association of a lincRNA-rs312691 variant with a genetic risk of TPP, an additional series of 37 patients with TPP and 32 patients with thyrotoxic without paralysis (TWP) were assessed. We verified that the risk of minor allele C was greater in TPP than in TWP (odds ratio, 5.289; P = 0.0062), and protective major allele T was more frequent than observed in the 1000 genome controls (odds ratio, 11.90; P < 0.0001). AS-KCNJ2 was downregulated during thyrotoxicosis in the TWP controls carrying allele T and were upregulated in those with TPP with risk allele C. Moreover, KCNJ2 (Kir2.1) expression was reduced during thyrotoxicosis and restored in euthyroid status. We further excluded any other coding variant by performing targeted exome sequencing mutational screening in 17q24.3. Our data suggest that high lincRNA AS-KCNJ2 and CDT-2378E21.1 expression, possibly driven by the triiodothyronine regulatory mechanism, reduces the Kir2.1 expression observed during thyrotoxicosis. This finding could contribute to the understanding of the reduced inward-rectifying current observed during muscle weakness in genetically susceptible TPP patients.

DUOX2 Mutations Are Associated With Congenital Hypothyroidism With Ectopic Thyroid Gland.

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.

Analysis of somatic mutations in BRAF, CDKN2A/p16 and PI3KCA in patients with medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), a neuroendocrine tumor originating from thyroid parafollicular cells, has been demonstrated to be associated with mutations in RET, HRAS, KRAS and NRAS. However, the role of other genes involved in the oncogenesis of neural crest tumors remains to be fully investigated in MTC. The current study aimed to investigate the presence of somatic mutations in BRAF, CDKN2A and PI3KCA in MTC, and to investigate the correlation with disease progression. DNA was isolated from paraffin­embedded tumors and blood samples from patients with MTC, and the hotspot somatic mutations were sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn and p.His94Tyr, and polymorphisms within the 3' untranslated region (UTR) of CDKN2A (rs11515 and rs3088440) were identified, however, no mutations were observed in other genes. It was suggested that somatic point mutations in BRAF, CDKN2A and PI3KCA do not participate in the oncogenesis of MTC. Further studies are required in order to clarify the contribution of the polymorphisms identified in the 3'UTR of CDKN2A in MTC.

Daily exposure to silver nanoparticles during prepubertal development decreases adult sperm and reproductive parameters.

As silver nanoparticles (AgNPs) have antimicrobial properties and potentiate the activity of some antibiotics, they are broadly used in both medical and nonmedical applications. In this study, prepubertal male Wistar rats were orally treated with 15 or 30 µg/kg/day AgNPs from postnatal day 23 (PND23) to PND58 and sacrificed at PND102. The acrosome integrity, plasma membrane integrity, mitochondrial activity and morphological alterations of the sperm were analyzed. Sexual partner preference, sexual behavior and the serum concentrations of FSH, LH, testosterone and estradiol were also recorded. The results were evaluated following the appropriate statistical analyses, and differences among the groups were considered significant when p < 0.05. AgNPs reduced the acrosome and plasma membrane integrities, reduced the mitochondrial activity and increased the abnormalities of the sperm in both treatment groups. AgNP exposure also delayed the onset of puberty, although no changes in body growth were observed in either treatment group. The animals did not show changes in sexual behavior or serum hormone concentrations. This study shows for the first time that prepubertal exposure to AgNPs causes alterations in adult sperm parameters. Importantly, the sperm appeared to be more sensitive to the toxic effects of AgNPs and demonstrated adverse effects following exposure to lower doses. Consequently, the effects of AgNPs on sperm should be considered in order to establish safety limits for the use of these particles.

Adult exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of the hypothalamic-pituitary-testicular axis.

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 25mg 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.

The absence of mutations in homeobox candidate genes HOXA3, HOXB3, HOXD3 and PITX2 in familial and sporadic thyroid hemiagenesis.

BACKGROUND: The molecular mechanisms leading to the formation of the two thyroid symmetrical lobes, which are impaired in thyroid hemiagenesis (TH), are little known. OBJECTIVE: The aim of this work was to search for mutations in thyroid developmental candidate genes HOXA3, HOXB3, HOXD3 and PITX2. METHODS: Total DNA from peripheral blood was extracted and then the entire coding region of all these genes was amplified by polymerase chain reaction and direct sequencing. RESULTS: Herein we describe familial cases of TH in two generations (proband and his father), in addition to other two sporadic cases. We have found polymorphisms in the HOXB3 (rs2229304), HOXD3 (rs34729309, rs1051929, c.543-199G>T and c.543-34G>A; and a new synonymous variant, NP_008829.3:p.314;C>G) and PITX2 (c.45+76C>T) genes, but no deleterious mutations. CONCLUSION: These results suggest the existence of other left-right thyroid asymmetry candidate genes in humans such as classical Mendelian mutation-causing disease, as well as other etiopathogenic mechanisms such as epigenetic modifications, especially for sporadic hemiagenesis.

Optimizing nucleic acid extraction from thyroid fine-needle aspiration cells in stained slides, formalin-fixed/paraffin-embedded tissues, and long-term stored blood samples.

OBJECTIVE: Adequate isolation of nucleic acids from peripheral blood, fine-needle aspiration cells in stained slides, and fresh and formalin-fixed/paraffin-embedded tissues is crucial to ensure the success of molecular endocrinology techniques, especially when samples are stored for long periods, or when no other samples can be collected from patients who are lost to follow-up. Here, we evaluate several procedures to improve current methodologies for DNA (salting-out) and RNA isolation. MATERIALS AND METHODS: We used proteinase K treatment, heat shock, and other adaptations to increase the amount and quality of the material retrieved from the samples. RESULTS: We successfully isolated DNA and RNA from the samples described above, and this material was suitable for PCR, methylation profiling, real-time PCR and DNA sequencing. CONCLUSION: The techniques herein applied to isolate nucleic acids allowed further reliable molecular analyses.

Optimizing nucleic acid extraction from thyroid fine-needle aspiration cells in stained slides, formalin-fixed/paraffin-embedded tissues, and long-term stored blood samples / Otimização da extração de ácidos nucleicos de material de punção aspirativa por agulha fina de tiroide obtido de lâminas coradas, tecidos fixados em formalina e emblocados em parafina e amostras de sangue estocadas por longo período

OBJECTIVE: Adequate isolation of nucleic acids from peripheral blood, fine-needle aspiration cells in stained slides, and fresh and formalin-fixed/paraffin-embedded tissues is crucial to ensure the success of molecular endocrinology techniques, especially when samples are stored for long periods, or when no other samples can be collected from patients who are lost to follow-up. Here, we evaluate several procedures to improve current methodologies for DNA (salting-out) and RNA isolation. MATERIALS AND METHODS: We used proteinase K treatment, heat shock, and other adaptations to increase the amount and quality of the material retrieved from the samples. RESULTS: We successfully isolated DNA and RNA from the samples described above, and this material was suitable for PCR, methylation profiling, real-time PCR and DNA sequencing. CONCLUSION: The techniques herein applied to isolate nucleic acids allowed further reliable molecular analyses. Arq Bras Endocrinol Metab. 2012;56(9):618-26.

OBJETIVO: O isolamento adequado de ácidos nucleicos a partir de sangue periférico, lâmina corada de punção aspirativa por agulha fina, tecido fixado em formalina e emblocado em parafina e tecido fresco é fundamental para assegurar o sucesso de técnicas aplicadas em endocrinologia molecular, principalmente quando lidamos com amostras estocadas por longos períodos ou quando há impossibilidade de nova coleta de amostra de pacientes que perderam o seguimento. Neste trabalho, objetivamos otimizar as metodologias clássicas para a extração de DNA (salting-out) e RNA. MATERIAIS E MÉTODOS: Utilizamos proteinase K, choque térmico, dentre outras modificações, com o objetivo de aumentar a quantidade e a qualidade do material recuperado a partir das amostras descritas acima. RESULTADOS: Isolamos com sucesso DNA e RNA de tais amostras e o material obtido foi adequado para a realização de PCR, perfil de metilação, PCR em tempo real e sequenciamento de DNA. CONCLUSÃO: As técnicas aplicadas neste estudo para isolar ácidos nucleicos permitiram a realização posterior de análises moleculares consistentes e confiáveis. Arq Bras Endocrinol Metab. 2012;56(9):618-26.