Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 (WT1) gene.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor ß-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Gene expression signatures predict response to therapy with growth hormone.

Recombinant human growth hormone (r-hGH) is used as a therapeutic agent for disorders of growth including growth hormone deficiency (GHD) and Turner syndrome (TS). Treatment is costly and current methods to model response are inexact. GHD (n = 71) and TS patients (n = 43) were recruited to study response to r-hGH over 5 years. Analysis was performed using 1219 genetic markers and baseline (pre-treatment) blood transcriptome. Random forest was used to determine predictive value of transcriptomic data associated with growth response. No genetic marker passed the stringency criteria for prediction. However, we identified an identical set of genes in both GHD and TS whose expression could be used to classify therapeutic response to r-hGH with a high accuracy (AUC > 0.9). Combining transcriptomic markers with clinical phenotype was shown to significantly reduce predictive error. This work could be translated into a single genomic test linked to a prediction algorithm to improve clinical management. Trial registration numbers: NCT00256126 and NCT00699855.

Clinical, genetic, and structural basis of congenital adrenal hyperplasia due to 11ß-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1, a gene encoding 11ß-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11ß-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11ß-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11ß-hydroxylase deficiency CAH.

Gonadotrophin-mediated miRNA expression in testis at onset of puberty in rhesus monkey: predictions on regulation of thyroid hormone activity and DLK1-DIO3 locus.

Molecular mechanisms responsible for the initiation of primate spermatogenesis remain poorly characterized. Previously, 48 h stimulation of the testes of three juvenile rhesus monkeys with pulsatile LH and FSH resulted in down-regulation of a cohort of genes recognized to favor spermatogonia stem cell renewal. This change in genetic landscape occurred in concert with amplification of Sertoli cell proliferation and the commitment of undifferentiated spermatogonia to differentiate. In this report, the non-protein coding small RNA transcriptomes of the same testes were characterized using RNA sequencing: 537 mature micro-RNAs (miRNAs), 322 small nucleolar RNAs (snoRNAs) and 49 small nuclear RNAs (snRNAs) were identified. Pathway analysis of the 20 most highly expressed miRNAs suggested that these transcripts contribute to limiting the proliferation of the primate Sertoli cell during juvenile development. Gonadotrophin treatment resulted in differential expression of 35 miRNAs, 12 snoRNAs and four snRNA transcripts. Ten differentially expressed miRNAs were derived from the imprinted delta-like homolog 1-iodothyronine deiodinase 3 (DLK1-DIO3) locus that is linked to stem cell fate decisions. Four gonadotrophin-regulated expressed miRNAs were predicted to trigger a local increase in thyroid hormone activity within the juvenile testis. The latter finding leads us to predict that, in primates, a gonadotrophin-induced selective increase in testicular thyroid hormone activity, together with the established increase in androgen levels, at the onset of puberty is necessary for the normal timing of Sertoli cell maturation, and therefore initiation of spermatogenesis. Further examination of this hypothesis requires that peripubertal changes in thyroid hormone activity of the testis of a representative higher primate be determined empirically.

Near-adult height in male kidney transplant recipients started on growth hormone treatment in late puberty.

BACKGROUND: Growth retardation and its impact on adult height is considered to be one of the most common complications in patients with chronic kidney disease (CKD). Treatment with recombinant human growth hormone (rhGH) has been effective in improving growth in kidney transplantation (KTx) patients, but little data are available on adult height in patients who began rhGh treatment in late puberty. METHODS: Near-adult height was evaluated in 13 KTx patients treated with rhGH [growth hormone group (GHGr); dose 9.33 mg/m2 per week] for a period of at least 18 months. At initiation of rhGH treatment, testicular volume was >8 ml and serum testosterone was >1 ng/ml compared with the control group (CGr) of ten KTx patients who did not receive rHGH. All subjects were of similar chronological age and bone age and had similar creatinine clearance (CrCl) levels, cumulative corticoid dose, height standard deviation score (SDS), target height SDS, and target height:initial height at the beginning of the study. RESULTS: Near-adult height was significantly greater in the GHGr than in the CGr (-1.8 ± 0.8 vs. -2.9 ± 1.1; p = 0.018). The difference between initial height and near-adult height in the GHGr revealed a significant height gain (initial height -3.1 ± 1.1; near-adult height -1.8 ± 0.8 SDS, respectively; delta 1.2 ± 0.3; p = 0.021). The CrCl level was not significantly different between the GHGr and CGr at either at study initiation or when attaining near-adult height (p = 0.74 and p = 0.23, respectively). CONCLUSIONS: Treatment with rhGH was effective in improving adult height in KTx patients who began treatment in late puberty, without any effect on renal function.

The testicular transcriptome associated with spermatogonia differentiation initiated by gonadotrophin stimulation in the juvenile rhesus monkey (Macaca mulatta).

STUDY QUESTION: What is the genetic landscape within the testis of the juvenile rhesus monkey (Macaca mulatta) that underlies the decision of undifferentiated spermatogonia to commit to a pathway of differentiation when puberty is induced prematurely by exogenous LH and FSH stimulation? SUMMARY ANSWER: Forty-eight hours of gonadotrophin stimulation of the juvenile monkey testis resulted in the appearance of differentiating B spermatogonia and the emergence of 1362 up-regulated and 225 down-regulated testicular mRNAs encoding a complex network of proteins ranging from enzymes regulating Leydig cell steroidogenesis to membrane receptors, and from juxtacrine and paracrine factors to transcriptional factors governing spermatogonial stem cell fate. WHAT IS KNOWN ALREADY: Our understanding of the cell and molecular biology underlying the fate of undifferentiated spermatogonia is based largely on studies of rodents, particularly of mice, but in the case of primates very little is known. The present study represents the first attempt to comprehensively address this question in a highly evolved primate. STUDY DESIGN, SIZE, DURATION: Global gene expression in the testis from juvenile rhesus monkeys that had been stimulated with recombinant monkey LH and FSH for 48 h (N = 3) or 96 h (N = 4) was compared to that from vehicle treated animals (N = 3). Testicular cell types and testosterone secretion were also monitored. PARTICIPANTS/MATERIALS, SETTING, METHODS: Precocious testicular puberty was initiated in juvenile rhesus monkeys, 14-24 months of age, using a physiologic mode of intermittent stimulation with i.v. recombinant monkey LH and FSH that within 48 h produced 'adult' levels of circulating LH, FSH and testosterone. Mitotic activity was monitored by immunohistochemical assays of 5-bromo-2'-deoxyuridine and 5-ethynyl-2'-deoxyuridine incorporation. Animals were bilaterally castrated and RNA was extracted from the right testis. Global gene expression was determined using RNA-Seq. Differentially expressed genes (DEGs) were identified and evaluated by pathway analysis. mRNAs of particular interest were also quantitated using quantitative RT-PCR. Fractions of the left testis were used for histochemistry or immunoflouresence. MAIN RESULTS AND THE ROLE OF CHANCE: Differentiating type B spematogonia were observed after both 48 and 96 h of gonadotrophin stimulation. Pathway analysis identified five super categories of over-represented DEGs. Repression of GFRA1 (glial cell line-derived neurotrophic factor family receptor alpha 1) and NANOS2 (nanos C2HC-type zinc finger 2) that favor spermatogonial stem cell renewal was noted after 48 and 96 h of LH and FSH stimulation. Additionally, changes in expression of numerous genes involved in regulating the Notch pathway, cell adhesion, structural plasticity and modulating the immune system were observed. Induction of genes associated with the differentiation of spermatogonia stem cells (SOHLH1(spermatogenesis- and oogenesis-specific basic helix-loop-helix 1), SOHLH2 and KIT (V-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog)) was not observed. Expression of the gene encoding STRA8 (stimulated by retinoic acid 8), a protein generally considered to mark activation of retinoic acid signaling, was below our limit of detection. LARGE SCALE DATA: The entire mRNA data set for vehicle and gonadotrophin treated animals (N = 10) has been deposited in the GEO-NCBI repository (GSE97786). LIMITATIONS REASONS FOR CAUTION: The limited number of monkeys per group and the dilution of low abundance germ cell transcripts by mRNAs contributed from somatic cells likely resulted in an underestimation of the number of differentially expressed germ cell genes. WIDER IMPLICATIONS OF THE FINDINGS: The findings that expression of GDNF (a major promoter of spermatogonial stem cell renewal) was not detected in the control juvenile testes, expression of SOHLH1, SOHLH2 and KIT, promoters of spermatogonial differentiation in mice, were not up-regulated in association with the gonadotrophin-induced generation of differentiating spermatogonia, and that robust activation of the retinoic acid signaling pathway was not observed, could not have been predicted. These unexpected results underline the importance of non-human primate models in translating data derived from animal research to the human situation. STUDY FUNDING/COMPETING INTEREST(S): The work described was funded by NIH grant R01 HD072189 to T.M.P. P.A. was supported by an Endocrine Society Summer Research Fellowship Award and CONICET (Argentine Research Council), S.N. by a grant from Vali-e-Asr Reproductive Health Research Center of Tehran University of Medical Sciences (grant #24335-39-92) to Dr Batool Hosseini Rashidi, and M.P.H. by grants from the National Health and Medical Research Council of Australia, and the Victorian State Government's Operational Infrastructure Support Program. The authors have nothing to disclose.

Two novel heterozygous missense variations within the GLI2 gene in two unrelated Argentine patients.

Several heterozygous GLI2 gene mutations have been reported in patients with isolated GH deficiency (IGHD) or multiple pituitary hormone deficiency (MPHD) with or without other malformations. The primary aim of this study was to analyze the presence of GLI2 gene alterations in a cohort of patients with IGHD or MPHD and ectopic/absent posterior pituitary. The coding sequence and flanking intronic regions of GLI2 gene were amplified and directly sequenced from gDNA of 18 affected subjects and relatives. In silico tools were applied to identify the functional impact of newly found variants (Polyphen2, SIFT, Mutation Taster). We identified two novel heterozygous missense variations in two unrelated patients, p.Arg231Gln and p.Arg226Leu, located in the repressor domain of the protein. Both variations affect highly conserved amino acids of the Gli2 protein and were not found in the available databases. In silico tools suggest that these variations might be disease causing. Our study suggests that the GLI2 gene may be one of the candidate genes to analyze when an association of pituitary hormone deficiency and developmental defects in posterior pituitary gland. The highly variable phenotype found suggests the presence of additional unknown factors that could contribute to the phenotype observed in these patients.

Three novel IGF1R mutations in microcephalic patients with prenatal and postnatal growth impairment.

BACKGROUND: IGF1R gene mutations have been associated with varying degrees of intrauterine and postnatal growth retardation, and microcephaly. OBJECTIVE: To identify and characterize IGF1R gene variations in a cohort of 28 Argentinean children suspected of having IGF-1 insensitivity, who were selected on the basis of the association of pre/postnatal growth failure and microcephaly. METHODS: The coding sequence and flanking intronic regions of IGF1R gene were amplified and directly sequenced. Functional characterization was performed by two in vitro assays: 1) [Methyl-(3) H] thymidine incorporation into DNA in fibroblast cell primary cultures from patients and controls treated with IGF-1 for 16-24 h. 2) PI3K/Akt pathway was evaluated with phospho-Akt (Ser473) STAR ELISA Kit (Millipore) in fibroblast cultures from patients and controls stimulated with IGF-1 for 10 min. Prepubertal clinical and GH-IGF-1 axis evaluation was followed up. RESULTS: We identified three novel heterozygous missense mutations in three unrelated patients, de novo p.Arg1256Ser, de novo p.Asn359Tyr and p.Tyr865Cys. In control cells, proliferation assay showed that IGF-1 significantly induced DNA synthesis at 20 h and Akt phosphorylation assay that it significantly stimulated phosphorylation after 10 min (P < 0·05 by anova and Bonferroni Tests). However, no significant increase was observed in any of the three patient fibroblasts in both functional studies. GH therapy growth response in two patients was inconsistent. CONCLUSION: These variations led to failure of the IGF1R function causing pre- and postnatal growth retardation and microcephaly. Microcephaly should be considered in the evaluation of SGA patients, because it seems to favour the frequency of detection of IGF1R mutations.

ERRγ1 and Aromatase Expression in Human Placental Tissues from Term Deliveries of Large for Gestational Age (LGA) Newborns.

INTRODUCTION: Being born either large (LGA) or small for gestational age (SGA) has been associated with an increased risk of developing metabolic syndrome in adulthood. However, the mechanism underlying this early programming remained unclear. Estrogen-related receptor gamma (ERRγ) is an orphan nuclear receptor with a high expression in human placenta, particularly ERRγ1. ERRγ has been proposed to play a central role in controlling genes involved in energy metabolism. In placenta, ERRγ1 acts as an oxygen-responsive transcription factor regulating aromatase (Aro) expression during trophoblast differentiation. Aromatase is an enzyme that catalyzes the synthesis of estrogens from androgens and is located in the syncytiotrophoblast. An adequate estrogen-androgen balance is required for normal pregnancy progression. Our aim was to analyze ERRγ1 and Aro mRNA in human placenta from term LGA newborns. We propose that ERRγ1 and CYP19A1 expressions in human placenta from LGA newborns are impaired, which would modify fetal programming of LGA newborns, since an imbalance in intrauterine estrogen-androgen ratio would be occurred Methods: Total RNA was obtained from placental tissues of LGA (GA: 39-41 weeks, n=8) and adequate for gestational age (AGA; 39-40 weeks, n=10) newborns. ERRγ1 and Aro mRNA variants were analyzed by RT2-PCR. Primers for Aro analysis were specific for Total aromatase (TotalAro) binding in exons 2-3 and for Active aromatase (ActAro) in exons 9-10. Aro protein was analyzed by Western-blot. RESULTS: ERRγ1 mRNA was significantly higher in LGA compare to AGA. TotalAro mRNA was significantly lower in LGA in comparison with AGA control. Similar results with Aro protein. In contrast ActAro/TotalAro ratio was higher in LGA compared to the AGA control. CONCLUSIONS: High expression of ERRγ1 as well as ActAro/TotalAro ratio in LGA suggests that ERRγ1 is involved in ActAro variant expression and hence disrupted estrogen-androgen balance in the intrauterine environment. We propose that dysregulation of ERRγ1 in placenta might modify the estrogen-androgen balance in the intrauterine environment in LGA newborns, possibly representing one of the key factors in the regulation of fetal programming.

Surgical treatment of secondary hyperparathyroidism in children with chronic kidney disease. Experience in 19 patients.

OBJECTIVES: Secondary hyperparathyroidism (sHPT) is an important contributor to bone disease and cardiovascular calcifications in children with chronic kidney disease (CKD). When conservative measures are ineffective, parathyroidectomy is indicated. The aim of our study was to evaluate the efficacy and safety of subtotal parathyroidectomy (sPTX) in pediatric and adolescent patients, and to provide a rationale for considering this aggressive treatment in CKD patients with uncontrolled sHPT. METHODS: We retrospectively analyzed the medical records of 19 pediatric CKD patients on dialysis with refractory sHPT who underwent sPTX at our institution between 2010 and 2020. All patients had clinical, radiological, and biochemical signs of renal osteodystrophy. RESULTS: One year after sPTX, parathyroid hormone (PTH) levels (median and interquartile range (IQR)) dropped from 2073 (1339-2484) to 164 (93-252) pg/mL (p=0.0001), alkaline phosphatase (ALP) levels from 1166 (764-2373) to 410 (126-421) IU/L (p=0.002), and the mean (±SDS) calcium-phosphate (Ca*P) product from 51±11 to 41±13 mg2/dL2 (p=0.07). Postoperatively, all patients presented with severe hungry bone syndrome (HBS) and required intravenous and oral calcium and calcitriol supplementation. None of them had other postoperative complication. Histological findings had a good correlation with preoperative parathyroid ultrasound imaging (n: 15) in 100 % and with technetium-99m (99mTc) sestamibi scintigraphy (n: 15) in 86.6 %. Clinical and radiological signs of bone disease improved in all patients. CONCLUSIONS: Pediatric sPTX is effective and safe to control sHPT and calcium-phosphate metabolism in children with CKD on dialysis and may mitigate irreversible bone deformities and progression of cardiovascular disease.

Exome Sequencing has a high diagnostic rate in sporadic congenital hypopituitarism and reveals novel candidate genes.

CONTEXT: The pituitary gland is key for childhood growth, puberty, and metabolism. Pituitary dysfunction is associated with a spectrum of phenotypes, from mild to severe. Congenital Hypopituitarism (CH) is the most commonly reported pediatric endocrine dysfunction with an incidence of 1:4000, yet low rates of genetic diagnosis have been reported. OBJECTIVE: We aimed to unveil the genetic etiology of CH in a large cohort of patients from Argentina. METHODS: We performed whole exome sequencing of 137 unrelated cases of CH, the largest cohort examined with this method to date. RESULTS: Of the 137 cases, 19.1% and 16% carried pathogenic or likely pathogenic variants in known and new genes, respectively, while 28.2% carried variants of uncertain significance. This high yield was achieved through the integration of broad gene panels (genes described in animal models and/or other disorders), an unbiased candidate gene screen with a new bioinformatics pipeline (including genes high loss of function intolerance), and analysis of copy number variants. Three novel findings emerged. First, the most prevalent affected gene encodes the cell adhesion factor ROBO1. Affected children had a spectrum of phenotypes, consistent with a role beyond pituitary stalk interruption syndrome. Second, we found that CHD7 mutations also produce a phenotypic spectrum, not always associated with full CHARGE syndrome. Third, we add new evidence of pathogenicity in the genes PIBF1 and TBC1D32, and report 13 novel candidate genes associated with CH (e.g. PTPN6, ARID5B). CONCLUSION: Overall, these results provide an unprecedented insight into the diverse genetic etiology of hypopituitarism.

[Novel heterozygous mutation in the steroidogenic acute regulatory protein gene in a 46,XY patient with congenital lipoid adrenal hyperplasia]. / Nueva mutación heterocigota en el gen de la proteína regulatoria aguda de la esteroideogénesis (StAR) en un paciente 46,XY con hiperplasia adrenal congénita lipoidea.

StAR facilitates cholesterol entry into the mitochondria as part of the transduceosome complex. Recessive mutations in the gen STAR cause classic and nonclassic congenital lipoid adrenal hyperplasia. The aim of the study was to analyze the molecular consequences of a novel heterozygous STAR mutation in a 46,XY patient with ambiguous genitalia and adrenal insufficiency. We found a de novo heterozygous IVS-2A>G STAR mutation and the reported heterozygous p.G146A SF1 polymorphism with normal CYP11A1, FDXR, FDX1, VDAC1 and TSPO genes. RT-PCR and sequencing from patient's testicular RNA showed a -exon2 transcript and the wild-type (WT) transcript. Both 37 kDa precursor and 30 kDa mature protein were detected in COS-7 cell transfected with mutant and WT plasmids. Immunofluorescence showed almost no co-localization of mitochondria and mutant protein (delta22-59StAR). Delta22-59StAR activity was 65±13% of WT. Cotransfection with WT and delta22-59StAR plasmids reduced WT activity by 62.0% ± 13.9. Novel splice-junction heterozygous STAR mutation (IVS-2A>G) resulted in the in-frame loss of amino acids 22 to 59 in the N-terminal mitochondrial targeting signal. A misfolded p.G22_L59delStAR might interfere with WT StAR activity by blocking the transduceosome complex, causing an autosomal dominant form of StAR deficiency, explaining the clinical phenotype.

A Surgical and Clinical Approach to Persistent Müllerian Duct Syndrome: Laparoscopic, Histological, and Molecular Findings.

BACKGROUND: Persistent müllerian duct syndrome (PMDS) is characterized by the persistence of müllerian duct derivatives in otherwise normally virilized 46,XY males. Biallelic mutations of the anti-müllerian hormone (AMH) and AMH receptor type 2 (AMHR2) genes lead to PMDS type 1 and 2, respectively. AIM: The aims of the study were to report the clinical, hormonal, and genetic findings in a patient with PMDS and discuss surgical strategies to achieve successful orchidopexy. RESULTS: A 4-year-old boy was evaluated after the incidental finding of müllerian derivates during laparoscopy for nonpalpable gonads. Karyotype was 46,XY and laboratory tests revealed normal serum gonadotropin and androgen levels but undetectable serum AMH levels. PMDS was suspected. Molecular analysis revealed a novel variant c.902_929del in exon 5 and a previously reported mutation (c.367C>T) in exon 1 of the AMH gene. Successful orchidopexy was performed in two sequential surgeries in which the müllerian duct structure was preserved and divided to protect the vascular supply to the gonads. Histological evaluation of the testicular biopsy showed mild signs of dysgenesis. Doppler ultrasound showed blood flow in both testes positioned in the scrotum 1.5 years after surgery. CONCLUSION: PMDS is a rare entity that requires a high index of suspicion (from surgeons) when evaluating a patient with bilateral cryptorchidism. Surgical treatment is challenging and long-term follow-up is essential. Histological evaluation of the testis deserves further investigation.

Age-specific thyroid hormone and thyrotropin reference intervals for a pediatric and adolescent population.

BACKGROUND: Establishment of reliable reference intervals remains valuable for confirming validity and advancing standardization across methods and populations. Moreover, knowledge of the measurement uncertainty (U) and of the reference change value (RCV) has important applications in clinical chemistry. METHODS: Starting from the information available in the laboratory data base (29,901 subjects) an initial selection was carried out by eliminating all subjects with a clinical or laboratory pathological report; data from 7581 0- to 20-year-old subjects (53.87% girls) remained in the study. These subjects, divided into nine age groups, were used to define reference distribution percentiles (2.5th, 50th and 97.5th) of serum thyrotropin (TSH), triiodothyronine (T3), thyroxine (T4), and free T4 (fT4), as well as U and RCV of these assays. RESULTS: In early infancy, T4 and fT4 values were higher than in the older age groups. Serum T4 95th percentile reference value, useful for the diagnosis of hyperthyroidism, was 142.9 in 20-year-old boys and 230.4 nmol/L in early infants and serum T3 95th percentile was 2.6 and 3.5 nmol/L, respectively, while fT4 2.5th percentile reference value, useful for the diagnosis of hypothyroidism, was 9.6 and 13.0 pmol/L, respectively. Serum TSH 97.5th percentile showed less age variation, 4.38-4.88 mIU/L. Performance of the four assays resulted in approximately 20% Us, reflecting simple and complex imprecision, trueness, analytical and functional sensitivity. RCV of serum TSH (58.6%) was larger than for thyroid hormones (28.3%-34.7%), probably due to the high biological variation of this hormone. CONCLUSIONS: We have established reference interval for TSH and thyroid hormones, as well as Us for assessing reliability of measurements, and RCVs to alert users on the presence of clinical significant changes.

Effectiveness of rhGH treatment on final height of renal-transplant recipients in childhood.

BACKGROUND: Growth retardation is a considerable clinical problem in children with chronic kidney disease (CKD). Optimization of metabolic and nutritional parameters does not always lead to improved growth. Recombinant human growth hormone (rhGH) treatment has been used to improve height. Several studies in the literature have shown increased growth velocity, although data on the final height (FH) reached are scarce. AIMS: We assessed the effect of rhGH on FH standard deviation score (SDS) in children with CKD following renal transplantation (RTx), comparing it with patients who did not receive rhGH (control group) but were treated with the same protocol and followed up in a single Center. METHODS: Thirty-three patients received rhGH treatment until FH. Fourteen who refused rhGH therapy were included in the controls. Prognostic factors for FH and changes in glomerular filtration rate (GFR) during follow-up were also analyzed RESULTS: FH SDS in rhGH-treated patients was significantly higher than in controls (-1.88 ± 1.14 vs -3.48 ± 1.19 SDS, respectively, p <0.05). In both groups, a similar reduction in GFR was observed. Height (SDS) at onset of rhGH treatment was the only statistically significant variable useful to predict response to treatment (p = 0.001). CONCLUSION: Our findings confirm that rhGH is effective to improve FH in CKD RTx patients, without affecting kidney function.

Congenital Adrenal Hyperplasia and Ehlers-Danlos Syndrome.

Congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase deficiency is an autosomal recessive disorder. The 21-hydroxylase enzyme P450c21 is encoded by the CYP21A2 gene located on chromosome 6p21.33 within the HLA major histocompatibility complex. This locus also contains the CYP21A1P, a non-functional pseudogene, that is highly homologous to the CYP21A2 gene. Other duplicated genes are C4A and C4B, that encode two isoforms of complement factor C4, the RP1 gene that encodes a serine/threonine protein kinase, and the TNXB gene that, encodes the extracellular matrix glycoprotein tenascin-X (TNX). TNX plays a role in collagen deposition by dermal fibroblasts and is expressed in the dermis of the skin and the connective tissue of the heart and skeletal muscle. During meiosis, misalignment may occur producing large gene deletions or gene conversion events resulting in chimeric genes. Chimeric recombination may occur between TNXB and TNXA. Three TNXA/TNXB chimeras have been described that differ in the junction site (CH1 to CH3) and result in a contiguous CYP21A2 and TNXB gene deletion, causing CAH-X syndrome. TNXB deficiency is associated with Ehlers Danlos syndrome (EDS). EDS comprises a clinically and genetically heterogeneous group of connective tissue disorders. As molecular analysis of the TNXB gene is challenging, the TNX-deficient type EDS is probably underdiagnosed. In this minireview, we will address the different strategies of molecular analysis of the TNXB-gene, as well as copy number variations and genetic status of TNXB in different cohorts. Furthermore, clinical features of EDS and clinical recommendations for long-term follow-up are discussed.

Pediatric adrenocortical tumors cohort characteristics and long-term follow-up at a single Argentinian tertiary center.

Pediatric adrenocortical tumors are rare and heterogeneous endocrine malignancies. OBJECTIVES: To report clinical, biochemical, and histological features, staging, and therapeutic interventions in a cohort of 28 patients treated at a single tertiary center. METHODS: A retrospective review of medical records of children with PACT (diagnosed before <18 years of age) followed between 1987-2018 at Hospital de Pediatría Garrahan, Buenos Aires, Argentina. RESULTS: Mean age at diagnosis was 4.6 years (range, 0.3-17.3 years) and median follow-up was 4.17 years (range, 0-12 years). Female to male ratio was 2.5:1. Signs and symptoms that prompted medical intervention were hormonal overproduction (57%), abdominal complaints (36%), and hypertensive encephalopathy (7%). In patients with clinically virilizing tumors (n=16) mean height standard deviation score (SDS) and bone age advance were significantly higher while body mass index (BMI) SDS was significantly lower than in those with clinical Cushing's (n=10) (p<0.05). Serum dehydroepiandrosterone sulfate (DHEAS) levels were significantly higher in stage IV than in stage I (p=0.03). Total adrenalectomy was performed in 26 patients. Eight patients (stage III-IV) received adjuvant chemotherapy. Five-year overall and disease-free survival were 100% for ST I-II, and 51% (95% CI 21-82) and 33% (95% CI 1.2-65) for ST III-IV, respectively (p=0.002). No statistical difference was found when comparing 2-year parameters with and without adjuvant chemotherapy. CONCLUSIONS: Height SDS and BMI SDS seem to mirror hormonal secretion in pediatric adrenocortical tumors. Higher DHEAS levels were found in patients with more advanced disease. Further large-scale studies are needed to validate a possible role for DHEAS as a biochemical marker of tumor stage and to draw robust conclusions on the use of adjuvant chemotherapy.

Gonadal tumor development in 46,XX disorders of gonadal development.

Background: Differences/disorders of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. Objective: The aim of this study is to report the histological characteristics and immunoexpression patterns of gonadal parenchyma in patients with 46,XX testicular and ovotesticular DSD, with a focus on the detection of germ cell malignancies. Design: Inclusion criteria were SRY-negative 46,XX testicular and ovotesticular DSD with available samples from gonadal biopsy or gonadectomy for the review of histological findings. Gonadal histology was assessed on hematoxylin and eosin-stained sections and immunohistochemical analysis. Histopathological criteria from the last World Health Organization classification of urogenital tumors were used to identify undifferentiated gonadal tissue, gonadoblastoma, and dysgerminoma. Results: Median age at first histological evaluation of gonadal samples was 1.46 years (range: 0.16-16 years). Totally 15 patients were classified as ovotesticular and only 1 as testicular DSD. Most individuals had bilateral ovotestes (12/15). No histological alterations were found in the ovarian parenchyma, while signs of dysgenesis were seen in all cases of testicular parenchyma. In 4/15 ovotesticular DSD, a prepubertal biopsy failed to identify ovarian parenchyma. We detected early prepubertal preinvasive and invasive malignancies in this cohort (five patients had undifferentiated gonadal tissue, five gonadoblastoma, and one dysgerminoma). Conclusion: 46,XX disorders of gonadal development are historically considered at a low risk for germ cell cancer, and the need for assessment of gonadal histology has been questioned. The finding of early germ cell malignancies in our cohort brings awareness and needs further research.

Histidine decarboxylase inhibitors: a novel therapeutic option for the treatment of leydigioma.

Recent reports indicate an increase in Leydig cell tumor (LCT) incidence. Radical orchiectomy is the standard therapy in children and adults, although it entails physical and psychosocial side effects. Testis-sparing surgery can be a consideration for benign LCT of 2.5 cm or less in size. Malignant LCTs respond poorly to conventional chemotherapy, so new treatment modalities are needed. In this study, we observed increased histidine decarboxylase expression and pro-angiogenic potential in LCT surgically resected from pediatric patients (fetal to pubertal) vs control samples from patients without endocrine or metabolic disorders which were collected at necropsy. We, therefore, evaluated for the first time the antitumor efficacy of two histidine decarboxylase inhibitors (α-methyl-dl-histidine dihydrochloride (α-MHD) and epigallocatechin gallate (EGCG)), alone and combined with carboplatin, in two preclinical models of LCT. MA-10 and R2C Leydig tumor cells, representing two different LCT subtypes, were used to generate syngeneic and xenograft mouse LCT models, respectively. In the syngeneic model, monotherapy with α-MHD effectively reduced tumor growth and angiogenesis. In the xenografts, which showed co-expression of histidine decarboxylase and CYP19, the combination of EGCG plus carboplatin was the most effective therapy, leading to LCT growth arrest and undetectable levels of plasmatic estradiol. Testicular and body weights remained unaltered. On the basis of this study, histidine decarboxylase may emerge as a novel pharmacological target for LCT treatment.