Publications by authors named "Antonio M Lerario"

58 Publications

Targeted massively parallel sequencing for congenital generalized lipodystrophy.

Arch Endocrinol Metab 2021 May;64(5):559-566

Grupo de Diabetes Monogênico, Unidade de Endocrinologia Genética/Laboratório de Investigação Médica (LIM/25) e Unidade de Diabetes, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil,

Objective: Our aim is to establish genetic diagnosis of congenital generalized lipodystrophy (CGL) using targeted massively parallel sequencing (MPS), also known as next-generation sequencing (NGS).

Methods: Nine unrelated individuals with a clinical diagnosis of CGL were recruited. We used a customized panel to capture genes related to genetic lipodystrophies. DNA libraries were generated, sequenced using the Illumina MiSeq, and bioinformatics analysis was performed.

Results: An accurate genetic diagnosis was stated for all nine patients. Four had pathogenic variants in and three in . Three large homozygous deletions in were identified by copy-number variant analysis.

Conclusion: Although we have found allelic variants in only 2 genes related to CGL, the panel was able to identify different variants including deletions that would have been missed by Sanger sequencing. We believe that MPS is a valuable tool for the genetic diagnosis of multi-genes related diseases, including CGL.
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http://dx.doi.org/10.20945/2359-3997000000278DOI Listing
May 2021

The phenotypic spectrum associated with OTX2 mutations in humans.

Eur J Endocrinol 2021 05 25;185(1):121-135. Epub 2021 May 25.

Section of Molecular Basis of Rare Disease, Genetics and Genomic Medicine Research & Teaching Department, UCL Great Ormond Street Institute of Child Health, London, UK.

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.
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http://dx.doi.org/10.1530/EJE-20-1453DOI Listing
May 2021

Significance of Alpha-inhibin Expression in Pheochromocytomas and Paragangliomas.

Am J Surg Pathol 2021 Apr 8. Epub 2021 Apr 8.

Department of Pathology, University Health Network Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Endocrine Oncology Site, The Princess Margaret Cancer Centre Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes Department of Pathology, University of Michigan, Ann Arbor, MI Department of Pathology, University Hospitals Cleveland Medical Center and Case Western Reserve University, Cleveland, OH.

Alpha-inhibin expression has been reported in pheochromocytomas and paragangliomas (PPGLs). We analyzed alpha-inhibin immunohistochemistry in 77 PPGLs (37 pheochromocytomas [PCCs] and 40 paragangliomas) and correlated the results with catecholamine profile, tumor size, Ki-67 labeling index, succinate dehydrogenase B subunit and carbonic anhydrase IX (CAIX) staining, and genetic pathogenesis. PPGLs were classified as pseudohypoxic cluster 1 disease with documented VHL mutation or SDHx mutation or biochemical phenotype, whereas NF1-driven and RET-driven PPGLs and those with a mature secretory (adrenergic or mixed adrenergic and noradrenergic) phenotype were classified as cluster 2 disease. The Cancer Genome Atlas data on INHA expression in PPGLs was examined. Alpha-inhibin was positive in 43 PPGLs (56%). Ki-67 labeling indices were 8.07% and 4.43% in inhibin-positive and inhibin-negative PPGLs, respectively (P<0.05). Alpha-inhibin expression did not correlate with tumor size. Alpha-inhibin was expressed in 92% of SDHx-related and 86% of VHL-related PPGLs. CAIX membranous staining was found in 8 of 51 (16%) tumors, including 1 SDHx-related PCC and all 5 VHL-related PCCs. NF1-driven and RET-driven PPGLs were negative for alpha-inhibin and CAIX. Alpha-inhibin was expressed in 77% of PPGLs with a pseudohypoxia signature, and 20% of PPGLs without a pseudohypoxia signature (P<0.05). PPGLs with a mature secretory phenotype were negative for CAIX. The Cancer Genome Atlas data confirmed higher expression of INHA in cluster 1 than in cluster 2 PPGLs. This study identifies alpha-inhibin as a highly sensitive (90.3%) marker for SDHx/VHL-driven pseudohypoxic PPGLs. Although CAIX has low sensitivity, it is the most specific biomarker of VHL-related pathogenesis. While alpha-inhibin cannot replace succinate dehydrogenase B subunit immunohistochemistry for detection of SDHx-related disease, it adds value in prediction of cluster 1 disease. Importantly, these data emphasize that alpha-inhibin is not a specific marker of adrenal cortical differentiation, as it is also expressed in PCCs.
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http://dx.doi.org/10.1097/PAS.0000000000001715DOI Listing
April 2021

Performance of mutation pathogenicity prediction tools on missense variants associated with 46,XY differences of sex development.

Clinics (Sao Paulo) 2021 22;76:e2052. Epub 2021 Jan 22.

Unidade de Endocrinologia do Desenvolvimento / LIM42 / SELA, Disciplina de Endocrinologia, Hospital das Clinicas (HCFMUSP), Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

Objectives: Single nucleotide variants (SNVs) are the most common type of genetic variation among humans. High-throughput sequencing methods have recently characterized millions of SNVs in several thousand individuals from various populations, most of which are benign polymorphisms. Identifying rare disease-causing SNVs remains challenging, and often requires functional in vitro studies. Prioritizing the most likely pathogenic SNVs is of utmost importance, and several computational methods have been developed for this purpose. However, these methods are based on different assumptions, and often produce discordant results. The aim of the present study was to evaluate the performance of 11 widely used pathogenicity prediction tools, which are freely available for identifying known pathogenic SNVs: Fathmn, Mutation Assessor, Protein Analysis Through Evolutionary Relationships (Phanter), Sorting Intolerant From Tolerant (SIFT), Mutation Taster, Polymorphism Phenotyping v2 (Polyphen-2), Align Grantham Variation Grantham Deviation (Align-GVGD), CAAD, Provean, SNPs&GO, and MutPred.

Methods: We analyzed 40 functionally proven pathogenic SNVs in four different genes associated with differences in sex development (DSD): 17β-hydroxysteroid dehydrogenase 3 (HSD17B3), steroidogenic factor 1 (NR5A1), androgen receptor (AR), and luteinizing hormone/chorionic gonadotropin receptor (LHCGR). To evaluate the false discovery rate of each tool, we analyzed 36 frequent (MAF>0.01) benign SNVs found in the same four DSD genes. The quality of the predictions was analyzed using six parameters: accuracy, precision, negative predictive value (NPV), sensitivity, specificity, and Matthews correlation coefficient (MCC). Overall performance was assessed using a receiver operating characteristic (ROC) curve.

Results: Our study found that none of the tools were 100% precise in identifying pathogenic SNVs. The highest specificity, precision, and accuracy were observed for Mutation Assessor, MutPred, SNP, and GO. They also presented the best statistical results based on the ROC curve statistical analysis. Of the 11 tools evaluated, 6 (Mutation Assessor, Phanter, SIFT, Mutation Taster, Polyphen-2, and CAAD) exhibited sensitivity >0.90, but they exhibited lower specificity (0.42-0.67). Performance, based on MCC, ranged from poor (Fathmn=0.04) to reasonably good (MutPred=0.66).

Conclusion: Computational algorithms are important tools for SNV analysis, but their correlation with functional studies not consistent. In the present analysis, the best performing tools (based on accuracy, precision, and specificity) were Mutation Assessor, MutPred, and SNPs&GO, which presented the best concordance with functional studies.
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http://dx.doi.org/10.6061/clinics/2021/e2052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811835PMC
April 2021

Late p65 nuclear translocation in glioblastoma cells indicates non-canonical TLR4 signaling and activation of DNA repair genes.

Sci Rep 2021 Jan 14;11(1):1333. Epub 2021 Jan 14.

Laboratory of Molecular and Cellular Biology (LIM15), Department of Neurology, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, São Paulo, SP, Brazil.

Glioblastoma (GBM) is the most aggressive brain primary malignancy. Toll-like receptor 4 (TLR4) has a dual role in cell fate, promoting cell survival or death depending on the context. Here, we analyzed TLR4 expression in different grades of astrocytoma, and observed increased expression in tumors, mainly in GBM, compared to non-neoplastic brain tissue. TLR4 role was investigated in U87MG, a GBM mesenchymal subtype cell line, upon LPS stimulation. p65 nuclear translocation was observed in late phase, suggesting TLR4-non-canonical pathway activation. In fact, components of ripoptosome and inflammasome cascades were upregulated and they were significantly correlated in GBMs of the TCGA-RNASeq dataset. Moreover, an increased apoptotic rate was observed when the GBM-derived U87MG cells were co-treated with LPS and Temozolomide (TMZ) in comparison to TMZ alone. Increased TLR4 immunostaining was detected in nuclei of U87MG cells 12 h after LPS treatment, concomitant to activation of DNA repair genes. Time-dependent increased RAD51, FEN1 and UNG expression levels were confirmed after LPS stimulation, which may contribute to tumor cell fitness. Moreover, the combined treatment with the RAD51 inhibitor, Amuvatinib in combination with, TMZ after LPS stimulation reduced tumor cell viability more than with each treatment alone. In conclusion, our results suggest that stimulation of TLR4 combined with pharmacological inhibition of the DNA repair pathway may be an alternative treatment for GBM patients.
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http://dx.doi.org/10.1038/s41598-020-79356-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809124PMC
January 2021

Comprehensive Genetic Analysis of 128 Candidate Genes in a Cohort With Idiopathic, Severe, or Familial Osteoporosis.

J Endocr Soc 2020 Dec 7;4(12):bvaa148. Epub 2020 Oct 7.

Laboratorio de Endocrinologia Celular e Molecular LIM-25, Divisao de Endocrinologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.

Context: The genetic bases of osteoporosis (OP), a disorder with high heritability, are poorly understood at an individual level. Cases of idiopathic or familial OP have long puzzled clinicians as to whether an actionable genetic cause could be identified.

Objective: We performed a genetic analysis of 28 cases of idiopathic, severe, or familial osteoporosis using targeted massively parallel sequencing.

Design: Targeted sequencing of 128 candidate genes was performed using Illumina NextSeq. Variants of interest were confirmed by Sanger sequencing or SNP array.

Patients And Setting: Thirty-seven patients in an academic tertiary hospital participated (54% male; median age, 44 years; 86% with fractures), corresponding to 28 sporadic or familial cases.

Main Outcome Measure: The identification of rare stop-gain, indel, splice site, copy-number, or nonsynonymous variants altering protein function.

Results: Altogether, we identified 28 variants of interest, but only 3 were classified as pathogenic or likely pathogenic variants: COL1A2 p.(Arg708Gln), WNT1 p.(Gly169Asp), and IDUA p.(His82Gln). An association of variants in different genes was found in 21% of cases, including a young woman with severe OP bearing , , and variants. Among genes of uncertain significance analyzed, a potential additional line of evidence has arisen for GWAS candidates and , warranting further studies.

Conclusions: While we hope that continuing efforts to identify genetic predisposition to OP will lead to improved and personalized care in the future, the likelihood of identifying actionable pathogenic variants in intriguing cases of idiopathic or familial osteoporosis is seemingly low.
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http://dx.doi.org/10.1210/jendso/bvaa148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645613PMC
December 2020

Phosphodiesterase 2A and 3B variants are associated with primary aldosteronism.

Endocr Relat Cancer 2021 01;28(1):1-13

Unidade de Suprarrenal, Laboratório de Hormônios e Genética Molecular LIM/42, Serviço de Endocrinologia e Metabologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.

Familial primary aldosteronism (PA) is rare and mostly diagnosed in early-onset hypertension (HT). However, 'sporadic' bilateral adrenal hyperplasia (BAH) is the most frequent cause of PA and remains without genetic etiology in most cases. Our aim was to investigate new genetic defects associated with BAH and PA. We performed whole-exome sequencing (paired blood and adrenal tissue) in six patients with PA caused by BAH that underwent unilateral adrenalectomy. Additionally, we conducted functional studies in adrenal hyperplastic tissue and transfected cells to confirm the pathogenicity of the identified genetic variants. Rare germline variants in phosphodiesterase 2A (PDE2A) and 3B (PDE3B) genes were identified in three patients. The PDE2A heterozygous variant (p.Ile629Val) was identified in a patient with BAH and early-onset HT at 13 years of age. Two PDE3B heterozygous variants (p.Arg217Gln and p.Gly392Val) were identified in patients with BAH and HT diagnosed at 18 and 33 years of age, respectively. A strong PDE2A staining was found in all cases of BAH in zona glomerulosa and/or micronodules (that were also positive for CYP11B2). PKA activity in frozen tissue was significantly higher in BAH from patients harboring PDE2A and PDE3B variants. PDE2A and PDE3B variants significantly reduced protein expression in mutant transfected cells compared to WT. Interestingly, PDE2A and PDE3B variants increased SGK1 and SCNN1G/ENaCg at mRNA or protein levels. In conclusion, PDE2A and PDE3B variants were associated with PA caused by BAH. These novel genetic findings expand the spectrum of genetic etiologies of PA.
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http://dx.doi.org/10.1530/ERC-20-0384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757641PMC
January 2021

Screening of targeted panel genes in Brazilian patients with primary ovarian insufficiency.

PLoS One 2020 23;15(10):e0240795. Epub 2020 Oct 23.

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular/LIM42, Hospital das Clínicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brazil.

Primary ovarian insufficiency (POI) is a heterogeneous disorder associated with several genes. The majority of cases are still unsolved. Our aim was to identify the molecular diagnosis of a Brazilian cohort with POI. Genetic analysis was performed using a customized panel of targeted massively parallel sequencing (TMPS) and the candidate variants were confirmed by Sanger sequencing. Additional copy number variation (CNV) analysis of TMPS samples was performed by CONTRA. Fifty women with POI (29 primary amenorrhea and 21 secondary amenorrhea) of unknown molecular diagnosis were included in this study, which was conducted in a tertiary referral center of clinical endocrinology. A genetic defect was obtained in 70% women with POI using the customized TMPS panel. Twenty-four pathogenic variants and two CNVs were found in 48% of POI women. Of these variants, 16 genes were identified as BMP8B, CPEB1, INSL3, MCM9, GDF9, UBR2, ATM, STAG3, BMP15, BMPR2, DAZL, PRDM1, FSHR, EIF4ENIF1, NOBOX, and GATA4. Moreover, a microdeletion and microduplication in the CPEB1 and SYCE1 genes, respectively, were also identified in two distinct patients. The genetic analysis of eleven patients was classified as variants of uncertain clinical significance whereas this group of patients harbored at least two variants in different genes. Thirteen patients had benign or no rare variants, and therefore the genetic etiology remained unclear. In conclusion, next-generation sequencing (NGS) is a highly effective approach to identify the genetic diagnoses of heterogenous disorders, such as POI. A molecular etiology allowed us to improve the disease knowledge, guide decisions about prevention or treatment, and allow familial counseling avoiding future comorbidities.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240795PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584253PMC
December 2020

Targeted massively parallel sequencing for congenital generalized lipodystrophy.

Arch Endocrinol Metab 2020 Aug 28. Epub 2020 Aug 28.

Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil.

Objective: Our aim is to establish genetic diagnosis of congenital generalized lipodystrophy (CGL) using targeted massively parallel sequencing (MPS), also known as next-generation sequencing (NGS).

Subjects And Methods: Nine unrelated individuals with a clinical diagnosis of CGL were recruited. We used a customized panel to capture genes related to genetic lipodystrophies. DNA libraries were generated, sequenced using the Illumina MiSeq, and bioinformatics analysis was performed.

Results: An accurate genetic diagnosis was stated for all nine patients. Four had pathogenic variants in AGPAT2 and three in BSCL2. Three large homozygous deletions in AGPAT2 were identified by copy-number variant analysis.

Conclusions: Although we have found allelic variants in only 2 genes related to CGL, the panel was able to identify different variants including deletions that would have been missed by Sanger sequencing. We believe that MPS is a valuable tool for the genetic diagnosis of multi-genes related diseases, including CGL.
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http://dx.doi.org/10.20945/2359-3997000000278DOI Listing
August 2020

Targeted RNAseq of Formalin-Fixed Paraffin-Embedded Tissue to Differentiate Among Benign and Malignant Adrenal Cortical Tumors.

Horm Metab Res 2020 Aug 13;52(8):607-613. Epub 2020 Aug 13.

Michigan Center for Translational Pathology, Ann Arbor, Michigan, USA.

Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., , , etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors.
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http://dx.doi.org/10.1055/a-1212-8803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880170PMC
August 2020

Statins Reduce Intratumor Cholesterol Affecting Adrenocortical Cancer Growth.

Mol Cancer Ther 2020 09 16;19(9):1909-1921. Epub 2020 Jun 16.

Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende, Cosenza, Italy.

Mitotane causes hypercholesterolemia in patients with adrenocortical carcinoma (ACC). We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to estrogen receptor α (ERα) action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production, and ERα activity and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them , whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content, and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ERα function. A mitochondrial target of ERα, the respiratory complex IV (COXIV), was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. Additionally, simvastatin reduced tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ERα, COXIV expression and activity and increase terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. Collectively, these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production and inhibits mitochondrial respiratory chain-inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-1063DOI Listing
September 2020

Urinary Sediment Transcriptomic and Longitudinal Data to Investigate Renal Function Decline in Type 1 Diabetes.

Front Endocrinol (Lausanne) 2020 30;11:238. Epub 2020 Apr 30.

Laboratório de Carboidratos e Radioimunoensaio (LIM-18), Faculdade de Medicina, Hospital das Clinicas HCFMUSP, Universidade de São Paulo, São Paulo, Brazil.

Using a discovery/validation approach we investigated associations between a panel of genes selected from a transcriptomic study and the estimated glomerular filtration rate (eGFR) decline across time in a cohort of type 1 diabetes (T1D) patients. Urinary sediment transcriptomic was performed to select highly modulated genes in T1D patients with rapid eGFR decline (decliners) vs. patients with stable eGFR (non-decliners). The selected genes were validated in samples from a T1D cohort ( = 54, mean diabetes duration of 21 years, 61% women) followed longitudinally for a median of 12 years in a Diabetes Outpatient Clinic. In the discovery phase, the transcriptomic study revealed 158 genes significantly different between decliners and non-decliners. Ten genes increasingly up or down-regulated according to renal function worsening were selected for validation by qRT-PCR; the genes , and were confirmed as differentially expressed comparing decliners vs. non-decliners after adjustment for potential confounders. , and significantly modified the slope of eGFR in T1D patients across time. Eight genes identified as differentially expressed in the urinary sediment of T1D patients presenting different eGFR decline rates significantly increased the accuracy of predicted renal function across time in the studied cohort. These genes may be a promising way of unveiling novel mechanisms associated with diabetic kidney disease progression.
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http://dx.doi.org/10.3389/fendo.2020.00238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204506PMC
May 2021

Clinical and Molecular Description of 16 Families With Heterozygous IHH Variants.

J Clin Endocrinol Metab 2020 08;105(8)

Skeletal Dysplasia Multidisciplinary Unit (UMDE), Hospital Universitario La Paz, Madrid, Spain.

Context: Heterozygous variants in the Indian hedgehog gene (IHH) have been reported to cause brachydactyly type A1 and mild hand and feet skeletal anomalies with short stature. Genetic screening in individuals with short stature and mild skeletal anomalies has been increasing over recent years, allowing us to broaden the clinical spectrum of skeletal dysplasias.

Objective: The objective of this article is to describe the genotype and phenotype of 16 probands with heterozygous variants in IHH.

Patients And Methods: Targeted next-generation sequencing or Sanger sequencing was performed in patients with short stature and/or brachydactyly for which the genetic cause was unknown.

Results: Fifteen different heterozygous IHH variants were detected, one of which is the first reported complete deletion of IHH. None of the patients showed the classical phenotype of brachydactyly type A1. The most frequently observed clinical characteristics were mild to moderate short stature as well as shortening of the middle phalanx on the fifth finger. The identified IHH variants were demonstrated to cosegregate with the short stature and/or brachydactyly in the 13 probands whose family members were available. However, clinical heterogeneity was observed: Two short-statured probands showed no hand radiological anomalies, whereas another 5 were of normal height but had brachydactyly.

Conclusions: Short stature and/or mild skeletal hand defects can be caused by IHH variants. Defects in this gene should be considered in individuals with these findings, especially when there is an autosomal dominant pattern of inheritance. Although no genotype-phenotype correlation was observed, cosegregation studies should be performed and where possible functional characterization before concluding that a variant is causative.
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http://dx.doi.org/10.1210/clinem/dgaa218DOI Listing
August 2020

Growth and Clinical Characteristics of Children with Floating-Harbor Syndrome: Analysis of Current Original Data and a Review of the Literature.

Horm Res Paediatr 2019 12;92(2):115-123. Epub 2019 Nov 12.

Unidade de Endocrinologia Genetica, Laboratorio de Endocrinologia Celular e Molecular LIM25, Disciplina de Endocrinologia da Faculdade de Medicina da Universidade de Sao Paulo (FMUSP), Sao Paulo, Brazil,

Background: Floating-Harbor syndrome (FHS) is a rare condition characterized by dysmorphic facial features, short stature, and expressive language delay.

Objective: The aim of this study was to describe a cohort of patients with FHS and review the literature about the response to recombinant human growth hormone (rhGH) therapy.

Methods: Anthropometric and laboratory data from 7 patients with FHS were described. The molecular diagnosis was established by multigene analysis. Moreover, we reviewed the literature concerning patients with FHS treated with rhGH.

Results: All 7 patients were born small for gestational age. At first evaluation, 6 patients had a height standard deviation score (SDS) ≤-2 and 1 had short stature in relation to their target height. Bone age was usually delayed, which rapidly advanced during puberty. Nonspecific skeletal abnormalities were frequently noticed, and normal to elevated plasma IGF-I levels were observed in all except 1 patient with growth hormone deficiency. Information about 20 patients with FHS treated with rhGH was analyzed (4 from our cohort and 16 from the literature). The median height changes during the treatment period (approx. 2.9 years) were 1.1 SDS (range from -0.4 to 3.1). Nontreated patients had an adult height SDS of -4.1 ± 1.2 (n = 10) versus -2.6 ± 0.8 SDS (n = 7, p 0.012) for treated patients.

Conclusion: We observed a laboratory profile compatible with IGF-1 insensitivity in some patients with FHS. Nevertheless, our study suggests that children with FHS may be considered as candidates for rhGH therapy. Further studies are necessary to establish the real benefit and safety of rhGH therapy in these patients.
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http://dx.doi.org/10.1159/000503782DOI Listing
May 2020

New Insights Into Pheochromocytoma Surveillance of Young Patients With Missense Mutations.

J Endocr Soc 2019 Sep 2;3(9):1682-1692. Epub 2019 Jul 2.

Unidade de Suprarrenal, Laboratório de Hormônios e Genética Molecular LIM/42, Serviço de Endocrinologia e Metabologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brasil.

Context: Von Hippel-Lindau (VHL) disease is an autosomal dominant syndrome caused by germline mutations in the gene. Guidelines recommend pheochromocytoma (PHEO) biochemical screening should start at age 5 years.

Objective: Genotype-phenotype correlations in VHL, focusing on PHEO penetrance in children, were studied.

Design: We retrospectively evaluated 31 individuals (median age at diagnosis was 26 years) with diagnosed VHL disease.

Results: PHEO was diagnosed in six children with VHL. A large PHEO (5 cm) was detected in a 4-year-old boy with p.Gly114Ser mutation. PHEO penetrance was 55% starting at age 4 years. missense mutations were identified in 11 of 22 families (50%), frameshift mutations in four (18.2%), stop codon in three (13.6%), splicing site in two (9.1%), and large gene deletion in two (9.1%). The codon 167 (n = 10) was a hotspot for mutations and was significantly associated with PHEO (90% 38%; = 0.007). PHEOs and pancreatic neuroendocrine tumors (PNETs) were strongly associated with missense mutations compared with other mutations (89.5% 0% and 73.7% 16.7%; = 0.0001 and 0.002, respectively). In contrast, pancreatic cysts (91.7% 26.3%; = 0.0001), renal cysts (66.7% 26.3%; = 0.027), and central nervous system hemangioblastomas (91.7% 47.3%; = 0.012) were more frequent in VHL with nonmissense mutations.

Conclusion: missense mutations were highly associated with PHEO and PNETs. Our data support that in children with VHL harboring missense mutations, biochemical screening for PHEO should be initiated at diagnosis.
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http://dx.doi.org/10.1210/js.2019-00225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735756PMC
September 2019

Evaluation of SHOX defects in the era of next-generation sequencing.

Clin Genet 2019 09 4;96(3):261-265. Epub 2019 Jul 4.

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular/LIM42, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.

Short stature homeobox (SHOX) haploinsufficiency is a frequent cause of short stature. Despite advances in sequencing technologies, the identification of SHOX mutations continues to be performed using standard methods, including multiplex ligation-dependent probe amplification (MLPA) followed by Sanger sequencing. We designed a targeted panel of genes associated with growth impairment, including SHOX genomic and enhancer regions, to improve the resolution of next-generation sequencing for SHOX analysis. We used two software packages, CONTRA and Nexus Copy Number, in addition to visual analysis to investigate the presence of copy number variants (CNVs). We evaluated 15 patients with previously known SHOX defects, including point mutations, deletions and a duplication, and 77 patients with idiopathic short stature (ISS). The panel was able to confirm all known defects in the validation analysis. During the prospective evaluation, we identified two new partial SHOX deletions (one detected only by visual analysis), including an intragenic deletion not detected by MLPA. Additionally, we were able to determine the breakpoints in four cases. Our results show that the designed panel can be used for the molecular investigation of patients with ISS, and it may even detect CNVs in SHOX and its enhancers, which may be present in a significant fraction of patients.
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http://dx.doi.org/10.1111/cge.13587DOI Listing
September 2019

Genetic diagnosis of congenital hypopituitarism by a target gene panel: novel pathogenic variants in GLI2, OTX2 and GHRHR.

Endocr Connect 2019 May;8(5):590-595

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM42, Disciplina de Endocrinologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brasil.

Aim: Congenital hypopituitarism has an incidence of 1:3500-10,000 births and is defined by the impaired production of pituitary hormones. Early diagnosis has an impact on management and genetic counselling. The clinical and genetic heterogeneity of hypopituitarism poses difficulties to select the order of genes to analyse. The objective of our study is to screen hypopituitarism genes (candidate and previously related genes) simultaneously using a target gene panel in patients with congenital hypopituitarism.

Methods: Screening of 117 subjects with congenital hypopituitarism for pathogenic variants in 26 genes associated with congenital hypopituitarism by massively parallel sequencing using a customized target gene panel.

Results: We found three novel pathogenic variants in OTX2 c.295C>T:p.Gln99*, GLI2 c.1681G>T:p.Glu561* and GHRHR c.820_821insC:p.Asp274Alafs*113, and the previously reported variants in GHRHR c.57+1G>A and PROP1 [c.301_302delAG];[c.109+1G>A].

Conclusions: Our results indicate that a custom-designed panel is an efficient method to screen simultaneously variants of biological and clinical relevance for congenital GH deficiency. A genetic diagnosis was possible in 5 out of 117 (4%) patients of our cohort. We identified three novel pathogenic variants in GHRHR, OTX2 and GLI2 expanding the spectrum of variants associated with congenital hypopituitarism.
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http://dx.doi.org/10.1530/EC-19-0085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510710PMC
May 2019

CD99 Expression in Glioblastoma Molecular Subtypes and Role in Migration and Invasion.

Int J Mol Sci 2019 Mar 6;20(5). Epub 2019 Mar 6.

Laboratory of Molecular and Cellular Biology (LIM 15), Department of Neurology, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo 01246-903, Brazil.

Glioblastoma (GBM) is the most aggressive type of brain tumor, with an overall survival of 17 months under the current standard of care therapy. CD99, an over-expressed transmembrane protein in several malignancies, has been considered a potential target for immunotherapy. To further understand this potentiality, we analyzed the differential expression of its two isoforms in human astrocytoma specimens, and the CD99 involved signaling pathways in glioma model U87MG cell line. CD99 was also analyzed in GBM molecular subtypes. Whole transcriptomes by RNA-Seq of -siRNA, and functional in vitro assays in -shRNA, that are found in U87MG cells, were performed. Astrocytoma of different malignant grades and U87MG cells only expressed CD99 isoform 1, which was higher in mesenchymal and classical than in proneural GBM subtypes. Genes related to actin dynamics, predominantly to focal adhesion, and lamellipodia/filopodia formation were down-regulated in the transcriptome analysis, when was silenced. A decrease in tumor cell migration/invasion, and dysfunction of focal adhesion, were observed in functional assays. In addition, a striking morphological change was detected in -silenced U87MG cells, further corroborating CD99 involvement in actin cytoskeleton rearrangement. Inhibiting the overexpressed CD99 may improve resectability and decrease the recurrence rate of GBM by decreasing tumor cells migration and invasion.
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http://dx.doi.org/10.3390/ijms20051137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429353PMC
March 2019

Exome Sequencing Reveals the POLR3H Gene as a Novel Cause of Primary Ovarian Insufficiency.

J Clin Endocrinol Metab 2019 07;104(7):2827-2841

Unidade de Endocrinologia do Desenvolvimento, Laboratorio de Hormonios e Genetica Molecular/LIM42, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP, Brazil.

Context: Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI.

Objective: To investigate the genetic etiology of idiopathic POI.

Patients And Methods: We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology.

Results: A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3hD50G) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3hD50G female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3hD50G mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles.

Conclusion: Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.
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http://dx.doi.org/10.1210/jc.2018-02485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543511PMC
July 2019

Genetics of aldosterone-producing adenomas with pathogenic KCNJ5 variants.

Endocr Relat Cancer 2019 04 1;26(4):463-470. Epub 2019 Feb 1.

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, Michigan, USA.

Somatic variants in genes that regulate intracellular ion homeostasis have been identified in aldosterone-producing adenomas (APA). Although the mechanisms leading to an increased aldosterone production in APA cells has been well studied, the molecular events that cause cell proliferation and tumor formation are poorly understood. In the present study, we have performed whole exome sequencing (WES) to characterize the landscape of somatic alterations in a homogeneous series of APA with pathogenic KCNJ5 variants. In the WES analysis on eleven APA, 84 exonic somatic events were called by 3 different somatic callers. Besides the KCNJ5 gene, only two genes (MED13 and ZNF669) harbored somatic variants in more than one APA. Unlike adrenocortical carcinomas, no chromosomal instability was observed by the somatic copy-number alteration and loss of heterozygosity analyses. The estimated tumor purity ranged from 0.35 to 0.67, suggesting a significant proportion of normal cell infiltration. Based on the results of PureCN analysis, the KCNJ5 variants appear to be clonal. In conclusion, in addition to KCNJ5 somatic pathogenic variant, no significant somatic event that would obviously explain proliferation or tumor growth was observed in our homogeneous cohort of KCNJ5-mutated APA. The molecular mechanisms causing APA growth and tumorigenesis remain to be elucidated.
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http://dx.doi.org/10.1530/ERC-18-0364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869655PMC
April 2019

A ZNRF3-dependent Wnt/β-catenin signaling gradient is required for adrenal homeostasis.

Genes Dev 2019 02 28;33(3-4):209-220. Epub 2019 Jan 28.

Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, Michigan 48109, USA.

Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/β-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike β-catenin gain-of-function models, which induce high Wnt/β-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/β-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing β-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/β-catenin activation, which is regulated by ZNRF3.
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http://dx.doi.org/10.1101/gad.317412.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362817PMC
February 2019

Mutations in MAP3K1 that cause 46,XY disorders of sex development disrupt distinct structural domains in the protein.

Hum Mol Genet 2019 05;28(10):1620-1628

Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA.

Missense mutations in the gene, MAP3K1, are a common cause of 46,XY gonadal dysgenesis, accounting for 15-20% of cases [Ostrer, 2014, Disorders of sex development (DSDs): an update. J. Clin. Endocrinol. Metab., 99, 1503-1509]. Functional studies demonstrated that all of these mutations cause a protein gain-of-function that alters co-factor binding and increases phosphorylation of the downstream MAP kinase pathway targets, MAPK11, MAP3K and MAPK1. This dysregulation of the MAP kinase pathway results in increased CTNNB1, increased expression of WNT4 and FOXL2 and decreased expression of SRY and SOX9. Unique and recurrent pathogenic mutations cluster in three semi-contiguous domains outside the kinase region of the protein, a newly identified N-terminal domain that shares homology with the Guanine Exchange Factor (residues Met164 to Glu231), a Plant HomeoDomain (residues Met442 to Trp495) and an ARMadillo repeat domain (residues Met566 to Glu862). Despite the presence of the mutation clusters and clinical data, there exists a dearth of mechanistic insights behind the development imbalance. In this paper, we use structural modeling and functional data of these mutations to understand alterations of the MAP3K1 protein and the effects on protein folding, binding and downstream target phosphorylation. We show that these mutations have differential effects on protein binding depending on the domains in which they occur. These mutations increase the binding of the RHOA, MAP3K4 and FRAT1 proteins and generally decrease the binding of RAC1. Thus, pathologies in MAP3K1 disrupt the balance between the pro-kinase activities of the RHOA and MAP3K4 binding partners and the inhibitory activity of RAC1.
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http://dx.doi.org/10.1093/hmg/ddz002DOI Listing
May 2019

Multigene Sequencing Analysis of Children Born Small for Gestational Age With Isolated Short Stature.

J Clin Endocrinol Metab 2019 06;104(6):2023-2030

Unidade de Endocrinologia Genética, Laboratório de Endocrinologia Celular e Molecular LIM25, Disciplina de Endocrinologia da Faculdade de Medicina da Universidade de São Paulo, São Paulo CEP, Brazil.

Context: Patients born small for gestational age (SGA) who present with persistent short stature could have an underlying genetic etiology that will account for prenatal and postnatal growth impairment. We applied a unique massive parallel sequencing approach in cohort of patients with exclusively nonsyndromic SGA to simultaneously interrogate for clinically substantial genetic variants.

Objective: To perform a genetic investigation of children with isolated short stature born SGA.

Design: Screening by exome (n = 16) or targeted gene panel (n = 39) sequencing.

Setting: Tertiary referral center for growth disorders.

Patients And Methods: We selected 55 patients born SGA with persistent short stature without an identified cause of short stature.

Main Outcome Measures: Frequency of pathogenic findings.

Results: We identified heterozygous pathogenic or likely pathogenic genetic variants in 8 of 55 patients, all in genes already associated with growth disorders. Four of the genes are associated with growth plate development, IHH (n = 2), NPR2 (n = 2), SHOX (n = 1), and ACAN (n = 1), and two are involved in the RAS/MAPK pathway, PTPN11 (n = 1) and NF1 (n = 1). None of these patients had clinical findings that allowed for a clinical diagnosis. Seven patients were SGA only for length and one was SGA for both length and weight.

Conclusion: These genomic approaches identified pathogenic or likely pathogenic genetic variants in 8 of 55 patients (15%). Six of the eight patients carried variants in genes associated with growth plate development, indicating that mild forms of skeletal dysplasia could be a cause of growth disorders in this group of patients.
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http://dx.doi.org/10.1210/jc.2018-01971DOI Listing
June 2019

Germline mutation landscape of multiple endocrine neoplasia type 1 using full gene next-generation sequencing.

Eur J Endocrinol 2018 12;179(6):391-407

Unidade de Endocrinologia Genetica UEG, Laboratorio de Endocrinologia Celular e Molecular LIM-25, Disciplina de Endocrinologia.

Background Loss-of-function germline MEN1 gene mutations account for 75-95% of patients with multiple endocrine neoplasia type 1 (MEN1). It has been postulated that mutations in non-coding regions of MEN1 might occur in some of the remaining patients; however, this hypothesis has not yet been fully investigated. Objective To sequence for the entire MEN1 including promoter, exons and introns in a large MEN1 cohort and determine the mutation profile. Methods and patients A target next-generation sequencing (tNGS) assay comprising 7.2 kb of the full MEN1 was developed to investigate germline mutations in 76 unrelated MEN1 probands (49 familial, 27 sporadic). tNGS results were validated by Sanger sequencing (SS), and multiplex ligation-dependent probe amplification (MLPA) assay was applied when no mutations were identifiable by both tNGS and SS. Results Germline MEN1 variants were verified in coding region and splicing sites of 57/76 patients (74%) by both tNGS and SS (100% reproducibility). Thirty-eight different pathogenic or likely pathogenic variants were identified, including 13 new and six recurrent variants. Three large deletions were detected by MLPA only. No mutation was detected in 16 patients. In untranslated, regulatory or in deep intronic MEN1 regions of the 76 MEN1 cases, no point or short indel pathogenic variants were found in untranslated, although 33 benign/likely benign and three new VUS variants were detected. Conclusions Our study documents that point or short indel mutations in non-coding regions of MEN1 are very rare events. Also, tNGS proved to be a highly effective technology for routine genetic MEN1 testing.
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http://dx.doi.org/10.1530/EJE-18-0430DOI Listing
December 2018

A 46,XX testicular disorder of sex development caused by a Wilms' tumour Factor-1 (WT1) pathogenic variant.

Clin Genet 2019 01 28;95(1):172-176. Epub 2018 Oct 28.

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular/LIM42, Hospital das Clínicas, Disciplina de Endocrinologia e Metabologia, Faculdade de Medicina da Universidade de São Paulo, Brazil.

Molecular diagnosis is rarely established in 46,XX testicular (T) disorder of sex development (DSD) individuals with atypical genitalia. The Wilms' tumour factor-1 (WT1) gene is involved in early gonadal development in both sexes. Classically, WT1 deleterious variants are associated with 46,XY disorders of sex development (DSD) because of gonadal dysgenesis. We report a novel frameshift WT1 variant identified in an SRY-negative 46,XX testicular DSD girl born with atypical genitalia. Target massively parallel sequencing involving DSD-related genes identified a novel heterozygous WT1 c.1453_1456del; p.Arg485Glyfs*14 variant located in the fourth zinc finger of the protein which is absent in the population databases. Segregation analysis and microsatellite analysis confirmed the de novo status of the variant that is predicted to be deleterious by in silico tools and to increase WT1 target activation in crystallographic model. This novel and predicted activating frameshift WT1 variant leading to the 46,XX testicular DSD phenotype includes the fourth zinc-finger DNA-binding domain defects in the genetic aetiology of 46,XX DSD.
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http://dx.doi.org/10.1111/cge.13459DOI Listing
January 2019

Drug repurposing using high-throughput screening identifies a promising drug combination to treat adrenocortical carcinoma.

Oncotarget 2018 Sep 7;9(70):33245-33246. Epub 2018 Sep 7.

Endocrine Oncology, Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA.

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http://dx.doi.org/10.18632/oncotarget.26091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161802PMC
September 2018

11-Ketotestosterone Is the Dominant Circulating Bioactive Androgen During Normal and Premature Adrenarche.

J Clin Endocrinol Metab 2018 12;103(12):4589-4598

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan.

Context: Adrenarche refers to the rise of dehydroepiandrosterone sulfate (DHEA-S) associated with the development of a functional adrenal zona reticularis. Clinical features of adrenarche include onset of body odor, axillary hair, and pubic hair, which reflect increased androgen action. An early rise in adrenal androgens, or premature adrenarche (PremA), is a risk factor for adverse metabolic profiles in adolescence and adulthood. The bioactive androgens associated with adrenarche and PremA remain poorly understood. The adrenal gland is a potential source of testosterone (T) and the 11-oxygenated derivatives 11β-hydroxytestosterone (11OHT) and 11-ketotestosterone (11KT).

Objective: The objective of this study was to characterize the adrenal androgen biome contributing to adrenarche and PremA.

Participants And Methods: With the use of mass spectrometry, 19 steroids including the 11-oxygenated derivatives of T were measured in sera obtained from girls with PremA (n = 37; 4 to 7 years) and age-matched girls (n = 83; 4 to 10 years).

Results: In reference population girls, dehydroepiandrosterone, DHEA-S, androstenediol-3-sulfate, T, and 11KT all increased at the onset of adrenarche (6 to 8 years) and beyond (9 to 10 years) (P < 0.05 vs younger subjects 4 to 5 years). T, 11OHT, and 11KT were further elevated in PremA vs age-matched girls (P < 0.001). Circulating concentrations of 11KT during adrenarche and PremA exceeded those of T and 11OHT (11KT > T ≥ 11OHT). Androgen receptor activity and nuclear translocation studies demonstrated that 11KT is a potent androgen similar to T.

Conclusions: Our findings suggest that 11KT is the dominant bioactive androgen in children during adrenarche and PremA. Its androgenic capacity suggests that it may be responsible for the phenotypic changes seen in these phenomena.
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http://dx.doi.org/10.1210/jc.2018-00736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226603PMC
December 2018

Novel SUZ12 mutations in Weaver-like syndrome.

Clin Genet 2018 11 6;94(5):461-466. Epub 2018 Aug 6.

Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

SUZ12 is a core component of polycomb repressive complex 2 (PRC2) along with EZH2 and EED. Recently, germline mutations in the SUZ12, EZH2 and EED genes have been reported in Weaver syndrome (WS) or Weaver-like syndrome, suggesting a functional link between PRC2 deficits and WS. However, only one case of a SUZ12 mutation presenting with Weaver-like syndrome has been reported. Here, we report a missense and a frameshift mutation in SUZ12 (c.1797A>C; p.Gln599His and c.844_845del; p.Ala282Glnfs*7), both of which are novel, in two individuals. Their clinical features included postnatal overgrowth, increased bifrontal diameter, large ears, round face, horizontal chin crease and skeletal anomalies, but did not fulfill the WS diagnostic criteria. These data provide strong evidence that SUZ12 mutations cause Weaver-like syndrome.
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http://dx.doi.org/10.1111/cge.13415DOI Listing
November 2018

Two rare loss-of-function variants in the STAG3 gene leading to primary ovarian insufficiency.

Eur J Med Genet 2019 Mar 10;62(3):186-189. Epub 2018 Jul 10.

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular/LIM42, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Laboratório de Sequenciamento em Larga Escala (SELA), Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brazil. Electronic address:

Background/aim: Primary ovarian insufficiency (POI) is characterized by primary or secondary amenorrhea, infertility, low estradiol levels, and increased gonadotropin levels. Most cases of POI remain unsolved even after exhaustive investigation. Here, we performed a targeted massively parallel sequencing to identify the genetic diagnosis of primary ovarian insufficiency (POI) in a Brazilian patient.

Patient And Methods: An adopted 21-year-old Brazilian woman with isolated POI was selected. A custom SureSelect DNA target enrichment panel was designed and sequenced on an Illumina NextSeq 500 sequencer. The variants were confirmed using Sanger sequencing.

Results: Two rare heterozygous pathogenic variants in the STAG3 gene were identified in our patient. An unpublished 1-bp duplication c.291dupC (p.Asn98Glnfs*2) and one stop codon variant c.1950C > A (p.Tyr650*) were identified in the STAG3 gene. Both undescribed heterozygous variants were absent in the public databases [1000Genomes, Exome Aggregation Consortium (ExAC), National Heart, Lung, and Blood Institute Exome Variant Server (NHLBI/EVS), database of Single Nucleotide Polymorphisms (dbSNP), Genome Aggregation Database (gnomAD)], and Online Archive of Brazilian Mutations (ABraOM) databases. Moreover, neither heterozygous variants were found in 400 alleles from fertile Brazilian women screened by Sanger sequencing. The parents' DNA was not available to segregate these variants.

Conclusion: Our results suggested that POI is caused by pathogenic compound heterozygous variants in the STAG3 gene, supporting the key role of the STAG3 gene in the etiology of primary ovarian insufficiency.
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http://dx.doi.org/10.1016/j.ejmg.2018.07.008DOI Listing
March 2019

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells.

Brain 2018 08;141(8):2299-2311

Département de Génétique, Hôpital La Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.
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http://dx.doi.org/10.1093/brain/awy173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061686PMC
August 2018