Neonatal Hydrocolpos in Bardet-Biedl Syndrome due to a Novel Frameshift Indel in the BBS10 Gene.

INTRODUCTION: Hydrocolpos, a rare condition characterized by cystic dilatation of the vagina, can arise from various etiologies, including isolated imperforate hymen and vaginal atresia. Genetic conditions, such as Bardet-Biedl syndrome (BBS), may also manifest with hydrocolpos as part of urogenital malformations. METHODS: We present a case of neonatal hydrocolpos associated with BBS. Sequencing of 19 BBS genes was performed to elucidate the genetic basis of the syndrome. RESULTS: Genetic analysis revealed a novel frameshift indel variant (c.1543_1546dup p.Thr516Argfs*7) in the BBS10 gene. This finding expands the spectrum of BBS mutations and underscores the importance of genetic evaluation in patients with hydrocolpos, particularly when associated with additional clinical features suggestive of syndromic etiology. CONCLUSION: Pediatric urologists should maintain a high index of suspicion for underlying genetic conditions, including BBS, in neonates presenting with hydrocolpos, given the potential for more severe associated complications such as renal and retinal diseases, obesity, and polydactyly.

Hydropathic AF-2 variants in the androgen receptor gene among androgen insensitivity patients.

BACKGROUND: Androgen insensitivity syndrome (AIS) is a common condition among individuals with differences of sexual development (DSD) and results from germline allelic variants in the androgen receptor (AR) gene. Understanding the phenotypic consequences of AR allelic variants that disrupt the activation function 2 (AF2) region is essential to grasping its clinical significance. OBJECTIVES: This study aims to provide insights into the phenotypic characteristics and clinical impact of AR mutations affecting the AF2 region in AIS patients. We achieve this by reviewing reported AR variants in the AF2 region among individuals with AIS, including identifying a new phenotype associated with the c.2138T>C variant (p.Leu713Pro) in the AR gene. MATERIALS AND METHODS: We comprehensively reviewed AR variants within the AF2 region reported in AIS and applied molecular dynamics simulations to assess the impact of the p.Leu713Pro variant on protein dynamics. RESULTS: Our review of reported AR variants in the AF2 region revealed a spectrum of phenotypic outcomes in AIS patients. Molecular dynamics simulations indicated that the p.Leu713Pro variant significantly alters the local dynamics of the AR protein and disrupts the correlation and covariance between variables. DISCUSSION: The diverse phenotypic presentations observed among individuals with AR variants in the AF2 region highlight the complexity of AIS. The altered protein dynamics resulting from the p.Leu713Pro variant further emphasize the importance of the AF2 region in AR function. CONCLUSION: Our study provides valuable insights into AR mutations' phenotypic characteristics and clinical impact on the AF2 region in AIS. Moreover, the disruption of protein dynamics underscores the significance of the AF2 region in AR function and its role in the pathogenesis of AIS.

The lncRNAMALAT1-WTAP axis: a novel layer of EMT regulation in hypoxic triple-negative breast cancer.

Early metastatic disease development is one characteristic that defines triple-negative breast cancer (TNBC) as the most aggressive breast cancer (BC) subtype. Numerous studies have identified long non-coding RNAs (lncRNA) as critical players in regulating tumor progression and metastasis formation. Here, we show that MALAT1, a long non-coding RNA known to promote various features of BC malignancy, such as migration and neo angiogenesis, regulates TNBC cell response to hypoxia. By profiling MALAT1-associated transcripts, we discovered that lncRNA MALAT1 interacts with the mRNA encoding WTAP protein, previously reported as a component of the N6-methyladenosine (m6A) modification writer complex. In hypoxic conditions, MALAT1 positively regulates WTAP protein expression, which influences the response to hypoxia by favoring the transcription of the master regulators HIF1α and HIF1ß. Furthermore, WTAP stimulates BC cell migratory ability and the expression of N-Cadherin and Vimentin, hallmarks of epithelial-to-mesenchymal transition (EMT). In conclusion, this study highlights the functional axis comprising MALAT1 and WTAP as a novel prognostic marker of TNBC progression and as a potential target for the development of therapeutic approaches for TNBC treatment.

ID4-dependent secretion of VEGFA enhances the invasion capability of breast cancer cells and activates YAP/TAZ via integrin ß3-VEGFR2 interaction.

Understanding the mechanisms of breast cancer cell communication underlying cell spreading and metastasis formation is fundamental for developing new therapies. ID4 is a proto-oncogene overexpressed in the basal-like subtype of triple-negative breast cancer (TNBC), where it promotes angiogenesis, cancer stem cells, and BRACA1 misfunction. Here, we show that ID4 expression in BC cells correlates with the activation of motility pathways and promotes the production of VEGFA, which stimulates the interaction of VEGFR2 and integrin ß3 in a paracrine fashion. This interaction induces the downstream focal adhesion pathway favoring migration, invasion, and stress fiber formation. Furthermore, ID4/ VEGFA/ VEGFR2/ integrin ß3 signaling stimulates the nuclear translocation and activation of the Hippo pathway member's YAP and TAZ, two critical executors for cancer initiation and progression. Our study provides new insights into the oncogenic roles of ID4 in tumor cell migration and YAP/TAZ pathway activation, suggesting VEGFA/ VEGFR2/ integrin ß3 axis as a potential target for BC treatment.

Injury-experienced satellite cells retain long-term enhanced regenerative capacity.

BACKGROUND: Inflammatory memory or trained immunity is a recently described process in immune and non-immune tissue resident cells, whereby previous exposure to inflammation mediators leads to a faster and stronger responses upon secondary challenge. Whether previous muscle injury is associated with altered responses to subsequent injury by satellite cells (SCs), the muscle stem cells, is not known. METHODS: We used a mouse model of repeated muscle injury, in which intramuscular cardiotoxin (CTX) injections were administered 50 days apart in order to allow for full recovery of the injured muscle before the second injury. The effect of prior injury on the phenotype, proliferation and regenerative potential of satellite cells following a second injury was examined in vitro and in vivo by immunohistochemistry, RT-qPCR and histological analysis. RESULTS: We show that SCs isolated from muscle at 50 days post-injury (injury-experienced SCs (ieSCs)) enter the cell cycle faster and form bigger myotubes when cultured in vitro, compared to control SCs isolated from uninjured contralateral muscle. Injury-experienced SCs were characterized by the activation of the mTORC 1 signaling pathway, suggesting they are poised to activate sooner following a second injury. Consequently, upon second injury, SCs accumulate in greater numbers in muscle at 3 and 10 days after injury. These changes in SC phenotype and behavior were associated with accelerated muscle regeneration, as evidenced by an earlier appearance of bigger fibers and increased number of myonuclei per fiber at day 10 after the second injury. CONCLUSIONS: Overall, we show that skeletal muscle injury has a lasting effect on SC function priming them to respond faster to a subsequent injury. The ieSCs have long-term enhanced regenerative properties that contribute to accelerated regeneration following a secondary challenge.

Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

Homozygous CDH2 variant may be associated with hypopituitarism without neurological disorders.

Context: Congenital hypopituitarism is a genetically heterogeneous condition. Whole exome sequencing (WES) is a promising approach for molecular diagnosis of patients with this condition. Objectives: The aim of this study is to conduct WES in a patient with congenital hypopituitarism born to consanguineous parents, CDH2 screening in a cohort of patients with congenital hypopituitarism, and functional testing of a novel CDH2 variant. Design: Genomic DNA from a proband and her consanguineous parents was analyzed by WES. Copy number variants were evaluated. The genetic variants were filtered for population frequency (ExAC, 1000 genomes, gnomAD, and ABraOM), in silico prediction of pathogenicity, and gene expression in the pituitary and/or hypothalamus. Genomic DNA from 145 patients was screened for CDH2 by Sanger sequencing. Results: One female patient with deficiencies in growth hormone, thyroid-stimulating hormone, adrenocorticotropic hormone, luteinizing hormone, and follicle-stimulating hormone and ectopic posterior pituitary gland contained a rare homozygous c.865G>A (p.Val289Ile) variant in CDH2. To determine whether the p.Val289Ile variant in CDH2 affects cell adhesion properties, we stably transfected L1 fibroblast lines, labeled the cells with lipophilic dyes, and quantified aggregation. Large aggregates formed in cells expressing wildtype CDH2, but aggregation was impaired in cells transfected with variant CDH2 or non-transfected. Conclusion: A homozygous CDH2 allelic variant was found in one hypopituitarism patient, and the variant impaired cell aggregation function in vitro. No disease-causing variants were found in 145 other patients screened for CDH2 variants. Thus, CDH2 is a candidate gene for hypopituitarism that needs to be tested in different populations. Significance statement: A female patient with hypopituitarism was born from consanguineous parents and had a homozygous, likely pathogenic, CDH2 variant that impairs cell aggregation in vitro. No other likely pathogenic variants in CDH2 were identified in 145 hypopituitarism patients.

Targeted massively parallel sequencing panel to diagnose genetic endocrine disorders in a tertiary hospital.

OBJECTIVES: To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. MATERIAL AND METHODS: Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. RESULTS: Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. CONCLUSION: The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.

Anti­oncogenic and pro­myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma.

Insights into the molecular and cellular biology of embryonal rhabdomyosarcoma (ERMS), an aggressive paediatric tumour, are required in order to identify new targets for novel treatments that may benefit patients with this disease. The present study examined the functional effects of MKK3 and MKK6, two upstream kinases of p38, and found that the ectopic expression of MKK6 led to rapid p38 activation and the myogenic differentiation of ERMS cells, whereas MKK3 failed to induce differentiation, while maintaining the proliferation state. Myogenin and myosin heavy chain were induced in MKK6­overexpressing ERMS cells and were inhibited by the p38 inhibitor, SB203580. The expression of Myc and ERK­PO4 increased under the effect of SB203580, whereas it decreased in MKK6­overexpressing cells. AKT activation was part of the myogenic program triggered by MKK6 overexpression alone. To the best of our knowledge, the present study demonstrates, for the first time, that the endogenous MKK6 pathway may be recovered by MEK/ERK inhibition (U0126 and trametinib) and that it concomitantly induces the reversal of the oncogenic pattern and the induction of the myogenic differentiation of ERMS cell lines. The effects of MEK/ERK inhibitors markedly increase the potential clinical applications in ERMS, particularly on account of the MEK inhibitor­induced early MKK6/p38 axis activation and of their anti­oncogenic effects. The findings presented herein lend further support to the antitumour effects of MKK6; MKK6 may thus represent a novel target for advanced personalised treatments against ERMS.

MALAT1-dependent hsa_circ_0076611 regulates translation rate in triple-negative breast cancer.

Vascular Endothelial Growth Factor A (VEGFA) is the most commonly expressed angiogenic growth factor in solid tumors and is generated as multiple isoforms through alternative mRNA splicing. Here, we show that lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and ID4 (inhibitor of DNA-binding 4) protein, previously referred to as regulators of linear isoforms of VEGFA, induce back-splicing of VEGFA exon 7, producing circular RNA circ_0076611. Circ_0076611 is detectable in triple-negative breast cancer (TNBC) cells and tissues, in exosomes released from TNBC cells and in the serum of breast cancer patients. Circ_0076611 interacts with a variety of proliferation-related transcripts, included MYC and VEGFA mRNAs, and increases cell proliferation and migration of TNBC cells. Mechanistically, circ_0076611 favors the expression of its target mRNAs by facilitating their interaction with components of the translation initiation machinery. These results add further complexity to the multiple VEGFA isoforms expressed in cancer cells and highlight the relevance of post-transcriptional regulation of VEGFA expression in TNBC cells.

Novel OTX2 loss of function variant associated with congenital hypopituitarism without eye abnormalities.

OBJECTIVES: The normal development of the pituitary gland requires multiple induction signals and transcription factors encoded by more than 30 genes, including OTX2. OTX2 mutations have been described with eye abnormalities and variable congenital hypopituitarism, but rarely with hypopituitarism without ocular manifestations. CASE PRESENTATION: We report a girl with hypopituitarism associated with pituitary hypoplasia and pituitary stalk atrophy, without ocular manifestations. NGS revealed a novel heterozygous mutation in OTX2 c.426dupC:p.(Ser143Leufs*2). CONCLUSIONS: Mutations in the transcription factor OTX2 have been associated with ocular, craniofacial, and pituitary development anomalies. Here we describe a novel mutation in OTX2 associated with hypopituitarism without an ocular phenotype.

Contribution of Clinical and Genetic Approaches for Diagnosing 209 Index Cases With 46,XY Differences of Sex Development.

CONTEXT: Massively parallel sequencing (MPS) technologies have emerged as a first-tier approach for diagnosing several pediatric genetic syndromes. However, MPS has not been systematically integrated into the diagnostic workflow along with clinical/biochemical data for diagnosing 46,XY differences of sex development (DSD). OBJECTIVE: To analyze the contribution of phenotypic classification either alone or in association with genetic evaluations, mainly MPS, for diagnosing a large cohort of 46,XY DSD patients. DESIGN/PATIENTS: 209 nonsyndromic 46,XY DSD index cases from a Brazilian DSD center were included. Patients were initially classified into 3 subgroups according to clinical and biochemical data: gonadal dysgenesis (GD), disorders of androgen secretion/action, and DSD of unknown etiology. Molecular genetic studies were performed by Sanger sequencing and/or MPS. RESULTS: Clinical/biochemical classification into either GD or disorders of hormone secretion/action was obtained in 68.4% of the index cases. Among these, a molecular diagnosis was obtained in 36% and 96.5%, respectively. For the remainder 31.6% classified as DSD of clinically unknown etiology, a molecular diagnosis was achieved in 31.8%. Overall, the molecular diagnosis was achieved in 59.3% of the cohort. The combination of clinical/biochemical and molecular approaches diagnosed 78.9% of the patients. Clinical/biochemical classification matched with the genetic diagnosis in all except 1 case. DHX37 and NR5A1 variants were the most frequent genetic causes among patients with GD and DSD of clinical unknown etiology, respectively. CONCLUSIONS: The combination of clinical/biochemical with genetic approaches significantly improved the diagnosis of 46,XY DSD. MPS potentially decreases the complexity of the diagnostic workup as a first-line approach for diagnosing 46,XY DSD.

Non-Coding RNAs in the Crosstalk between Breast Cancer Cells and Tumor-Associated Macrophages.

Non-coding RNAs (ncRNAs) play a pivotal role in regulating the tumor microenvironment (TME) by controlling gene expression at multiple levels. In tumors, ncRNAs can mediate the crosstalk between cancer cells and other cells in the TME, such as immune cells, stromal cells, and endothelial cells, influencing tumor development and progression. Tumor-associated macrophages (TAMs) are among the most abundant inflammatory cells infiltrating solid cancers that promote tumorigenesis, and their infiltration correlates with a poor prognosis in many tumors. Cancer cells produce different ncRNAs that orchestrate TAM recruitment and polarization toward a tumor-promoting phenotype. Tumor-reprogrammed macrophages shape the TME by promoting angiogenesis and tissue remodeling, and suppressing the anti-tumor activity of adaptive immune cells. TAMs can also produce ncRNA molecules that boost cancer cell proliferation and direct their phenotype and metabolic changes facilitating cancer progression and metastasis. This review will focus on the crosstalk between cancer cells and TAMs mediated by microRNAs and long non-coding RNAs during breast cancer (BC) initiation and progression.

Cushing disease due to a somatic USP8 mutation in a patient with evolving pituitary hormone deficiencies due to a germline GH1 splicing variant

SUMMARY We present the unique case of an adult Brazilian woman with severe short stature due to growth hormone deficiency with a heterozygous G to T substitution in the donor splice site of intron 3 of the growth hormone 1 (GH1) gene (c.291+1G>T). In this autosomal dominant form of growth hormone deficiency (type II), exon 3 skipping results in expression of the 17.5 kDa isoform of growth hormone, which has a dominant negative effect over the bioactive isoform, is retained in the endoplasmic reticulum, disrupts the Golgi apparatus, and impairs the secretion of other pituitary hormones in addition to growth hormone deficiency. This mechanism led to the progression of central hypothyroidism in the same patient. After 5 years of growth and thyroid hormone replacement, at the age of 33, laboratory evaluation for increased weight gain revealed high serum and urine cortisol concentrations, which could not be suppressed with dexamethasone. Magnetic resonance imaging of the sella turcica detected a pituitary macroadenoma, which was surgically removed. Histological examination confirmed an adrenocorticotropic hormone (ACTH)-secreting pituitary macroadenoma. A ubiquitin-specific peptidase 8 (USP8) somatic pathogenic variant (c.2159C>G/p.Pro720Arg) was found in the tumor. In conclusion, we report progression of isolated growth hormone deficiency due to a germline GH1 variant to combined pituitary hormone deficiency followed by hypercortisolism due to an ACTH-secreting macroadenoma with a somatic variant in USP8 in the same patient. Genetic studies allowed etiologic diagnosis and prognosis of this unique case.

Cushing disease due to a somatic USP8 mutation in a patient with evolving pituitary hormone deficiencies due to a germline GH1 splicing variant.

We present the unique case of an adult Brazilian woman with severe short stature due to growth hormone deficiency with a heterozygous G to T substitution in the donor splice site of intron 3 of the growth hormone 1 (GH1) gene (c.291+1G>T). In this autosomal dominant form of growth hormone deficiency (type II), exon 3 skipping results in expression of the 17.5 kDa isoform of growth hormone, which has a dominant negative effect over the bioactive isoform, is retained in the endoplasmic reticulum, disrupts the Golgi apparatus, and impairs the secretion of other pituitary hormones in addition to growth hormone deficiency. This mechanism led to the progression of central hypothyroidism in the same patient. After 5 years of growth and thyroid hormone replacement, at the age of 33, laboratory evaluation for increased weight gain revealed high serum and urine cortisol concentrations, which could not be suppressed with dexamethasone. Magnetic resonance imaging of the sella turcica detected a pituitary macroadenoma, which was surgically removed. Histological examination confirmed an adrenocorticotropic hormone (ACTH)-secreting pituitary macroadenoma. A ubiquitin-specific peptidase 8 (USP8) somatic pathogenic variant (c.2159C>G/p.Pro720Arg) was found in the tumor. In conclusion, we report progression of isolated growth hormone deficiency due to a germline GH1 variant to combined pituitary hormone deficiency followed by hypercortisolism due to an ACTH-secreting macroadenoma with a somatic variant in USP8 in the same patient. Genetic studies allowed etiologic diagnosis and prognosis of this unique case.

Variants in 46,XY DSD-Related Genes in Syndromic and Non-Syndromic Small for Gestational Age Children with Hypospadias.

Hypospadias is a common congenital disorder of male genital formation. Children born small for gestational age (SGA) present a high frequency of hypospadias of undetermined etiology. No previous study investigated the molecular etiology of hypospadias in boys born SGA using massively parallel sequencing. Our objective is to report the genetic findings of a cohort of patients born SGA with medium or proximal hypospadias. We identified 46 individuals with this phenotype from a large cohort of 46,XY DSD patients, including 5 individuals with syndromic features. DNA samples from subjects were studied by either whole exome sequencing or target gene panel approach. Three of the syndromic patients have 5 main clinical features of Silver-Russell syndrome (SRS) and were first studied by MLPA. Among the syndromic patients, loss of DNA methylation at the imprinting control region H19/IGF2 was identified in 2 individuals with SRS clinical diagnosis. Two novel pathogenic variants in compound heterozygous state were identified in the CUL7 gene establishing the diagnosis of 3M syndrome in one patient, and a novel homozygous variant in TRIM37 was identified in another boy with Mulibrey nanism phenotype. Among the non-syndromic subjects, 7 rare heterozygous variants were identified in 6 DSD-related genes. However, none of the variants found can explain the phenotype by themselves. In conclusion, a genetic defect that clarifies the etiology of hypospadias was not found in most of the non-syndromic SGA children, supporting the hypothesis that multifactorial causes, new genes, and/or unidentified epigenetic defects may have an influence in this condition.

Targeted massively parallel sequencing panel to diagnose genetic endocrine disorders in a tertiary hospital

Abstract Objectives To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. Material and methods Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. Results Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. Conclusion The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.

Allelic Variants in Established Hypopituitarism Genes Expand Our Knowledge of the Phenotypic Spectrum.

We report four allelic variants (three novel) in three genes previously established as causal for hypopituitarism or related disorders. A novel homozygous variant in the growth hormone gene, GH1 c.171delT (p.Phe 57Leufs*43), was found in a male patient with severe isolated growth hormone deficiency (IGHD) born to consanguineous parents. A hemizygous SOX3 allelic variant (p.Met304Ile) was found in a male patient with IGHD and hypoplastic anterior pituitary. YASARA, a tool to evaluate protein stability, suggests that p.Met304Ile destabilizes the SOX3 protein (ΔΔG = 2.49 kcal/mol). A rare, heterozygous missense variant in the TALE homeobox protein gene, TGIF1 (c.268C>T:p.Arg90Cys) was found in a patient with combined pituitary hormone deficiency (CPHD), diabetes insipidus, and syndromic features of holoprosencephaly (HPE). This variant was previously reported in a patient with severe holoprosencephaly and shown to affect TGIF1 function. A novel heterozygous TGIF1 variant (c.82T>C:p.Ser28Pro) was identified in a patient with CPHD, pituitary aplasia and ectopic posterior lobe. Both TGIF1 variants have an autosomal dominant pattern of inheritance with incomplete penetrance. In conclusion, we have found allelic variants in three genes in hypopituitarism patients. We discuss these variants and associated patient phenotypes in relation to previously reported variants in these genes, expanding our knowledge of the phenotypic spectrum in patient populations.

The phenotypic spectrum associated with OTX2 mutations in humans.

Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development. Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Genetic and clinical aspects of paediatric pheochromocytomas and paragangliomas.

OBJECTIVE: Few and conflicting reports have characterized the genetics of paediatric pheochromocytomas and paragangliomas (PPGLs). This study aimed to investigate the clinical and genetic features of Brazilian children with PPGL. PATIENTS AND METHODS: This study included 25 children (52% girls) with PPGL. The median age at diagnosis was 15 years (4-19). The median time of follow-up was 145 months. The genetic investigation was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification and/or target next-generation sequencing panel. RESULTS: Of the 25 children with PPGL, 11 (44%), 4 (16%), 2 (8%), 1 (4%) and 7 (28%) had germline VHL pathogenic variants, SDHB, SDHD, RET and negative genetic investigation, respectively. Children with germline VHL missense pathogenic variants were younger than those with SDHB or SDHD genetic defects [median (range), 12 (4-16) vs. 15.5 (14-19) years; P = .027]. Moreover, 10 of 11 cases with VHL pathogenic variants had bilateral pheochromocytoma (six asynchronous and four synchronous). All children with germline SDHB pathogenic variants presented with abdominal paraganglioma (one of them malignant). The two cases with SDHD pathogenic variants presented with head and neck paraganglioma. Among the cases without a genetic diagnosis, 6 and 2 had pheochromocytoma and paraganglioma, respectively. Furthermore, metastatic PPGL was diagnosed in four (16%) of 25 PPGL. CONCLUSIONS: Most of the paediatric PPGL were hereditary and multifocal. The majority of the affected genes belong to pseudohypoxic cluster 1, with VHL being the most frequently mutated. Therefore, our findings impact surgical management and surveillance of children with PPGL.