María Gabriela Ropelato

Context: Survival after pediatric hematologic malignancies has improved substantially, increasing concern regarding endocrine sequelae. While gonadotoxic effects on germ cells are characterized in adult survivors, the early impact of cancer and chemotherapy on hypothalamic–pituitary–testicular axis during childhood and adolescence remains insufficiently studied. Design: We conducted a prospective longitudinal cohort study including 63 boys and adolescents with acute lymphoblastic leukemia, acute myeloid leukemia, or non-Hodgkin lymphoma treated at a tertiary pediatric center in Argentina between 2013 and 2019. Hormonal markers of Sertoli cell (AMH, inhibin B, FSH) and Leydig cell function (testosterone, LH) were serially assessed from diagnosis, throughout chemotherapy, and up to 3 years after treatment completion. Results: In prepubertal boys, Sertoli cell function was mildly impaired at diagnosis, with reduced AMH and FSH levels. AMH increased during induction chemotherapy, fluctuated during treatment, and normalized thereafter, remaining stable up to 3 years post-treatment. Transient FSH suppression coincided with exposure to high-dose corticosteroids. Boys entering puberty during follow-up showed persistently elevated AMH relative to pubertal stage, suggesting delayed Sertoli cell maturation. In pubertal patients, a transient seminiferous tubular dysfunction, reflected by increased FSH and decreased inhibin B occurred, with recovery by 3 years after treatment. Mild compensated Leydig cell dysfunction persisted, characterized by elevated LH despite normal testosterone concentrations. Conclusions: Testicular dysfunction associated with pediatric hematologic malignancies is mostly reversible. However, delayed Sertoli cell maturation and persistent compensated Leydig cell insufficiency indicate residual testicular vulnerability, underscoring the importance of long-term endocrine surveillance during and after the end of chemotherapy.

Complex genetic syndromes represent a diagnostic challenge due to their diverse phenotypic presentations, which often evolve over time and may not be fully evident at birth. Disorders of sex development (DSD) comprise congenital conditions with discordance between chromosomal, gonadal, and/or genital sex. In 46,XY gonadal dysgenesis, undervirilisation or female-appearing genitalia may occur despite a normal karyotype, and diagnosis increasingly relies on genomic approaches. Prenatal and postnatal growth failure has been described in patients with syndromic 46,XY DSD. We report a male patient with SGA, lack of postnatal catch-up growth, and syndromic dysgenetic 46,XY DSD followed longitudinally from infancy to 11 years, in whom whole-exome sequencing (WES) reanalysis revealed a pathogenic 2.7 Mb microdeletion at 3q27.1q27.2. Systematic review of previously reported 3q27.1 deletions identified overlapping phenotypes but limited documentation of gonadal dysfunction. Curation of 71 genes within the deleted region highlighted DVL3 and CLCN2 as potential contributors to the gonadal phenotype, although functional evidence remains lacking. This case expands the phenotypic spectrum of 3q27.1 microdeletion syndrome, suggesting that 46,XY gonadal dysgenesis may represent an under-recognised feature. It also underscores the importance of copy number variant (CNV) analysis and periodic re-evaluation of sequencing data to increase diagnostic yield.

There is growing interest in replacing steroid immunoassays with liquid chromatography–tandem mass spectrometry-based methods in many clinical and research applications. The aim of the present work is to develop and validate an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS)-based method using atmospheric pressure chemical ionisation (APCI) to quantify cortisol, androstenedione, testosterone, pregnenolone, progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, and dehydroepiandrosterone in paediatric serum samples. Sample preparation was performed by protein precipitation using 100 µL of sample. The analytical method was validated in accordance with FDA and EMA international guidelines. Commercial quality control materials and a reference material (NIST®SRM®1950) were analysed for external assessment. Clinical applicability was tested in 61 infants under three months of age (50 healthy infants, eight preterm neonates, and three patients with confirmed adrenal disorders). The LLOQs were lower than 8 nmol/L; within- and between-run CVs were <12 %. Accuracy, in terms of recovery, was 89–111 %. Serum cortisol levels varied widely in healthy infants, and only testosterone levels exhibited sexual dimorphism (p < 0.0001). Androstenedione and 17-hydroxyprogesterone levels were significantly higher in the preterm group compared with babies born at term. Patients with confirmed adrenal pathologies exhibited abnormal steroid profiles. A novel, accurate, and sensitive UHPLC-APCI-MS/MS-based method for the simultaneous analysis of eight steroids in serum was developed, validated and successfully applied to infants younger than three months of age. The method enabled detection of abnormal values of precursors and steroid hormones in patients with adrenal disorders, with the potential for a large impact on paediatric patient care.

Context: P450 oxidoreductase (POR) deficiency is a rare congenital adrenal hyperplasia with variable severity. The mechanisms of severe mutations and their full metabolic consequences, including drug metabolism, are not fully characterized. Objective: To define the clinical, biochemical, and molecular consequences of a novel homozygous POR missense mutation, p.Gly88Ser (G88S), identified in 4 unrelated Argentine families. Design: A translational study combining clinical case series analysis with comprehensive in vitro molecular and functional characterization of the novel protein variant. Setting: Tertiary pediatric endocrine centers in Argentina and Switzerland. Patients: We report 5 individuals (4 46,XY; 1 46,XX) from 4 unrelated families presenting with disorders of sex development and adrenal dysfunction. Main Outcome Measures: Clinical phenotypes, hormonal profiles, and POR gene sequencing. In vitro analysis of recombinant POR measured flavin content, reductase activity, and support of steroidogenic and drug-metabolizing P450s. Results: All patients were homozygous for the c.262G>A (p.G88S) mutation. This FMN binding domain variant caused protein instability with severe loss of FMN (<30%) and FAD (<15%) cofactors. Steroidogenic activities were virtually abolished (CYP21A2: 1.3%; CYP17A1 17,20-lyase: 5.5% of wild-type), explaining the clinical phenotype. Activities of major drug-metabolizing enzymes were also severely impaired (3%-9% of wild-type), establishing a “poor metabolizer” phenotype. Conclusion: The POR G88S mutation causes one of the most severe forms of PORD described, driven by dynamic protein instability and cofactor loss. It is a critical pharmacogenomic marker, and its recurrence in Argentina suggests a potential screening target.

Context: P450 oxidoreductase (POR) deficiency is a rare congenital adrenal hyperplasia with variable severity. The mechanisms of severe mutations and their full metabolic consequences, including drug metabolism, are not fully characterized. Objective: To define the clinical, biochemical, and molecular consequences of a novel homozygous POR missense mutation, p.Gly88Ser (G88S), identified in four unrelated Argentine families. Design: A translational study combining clinical case series analysis with comprehensive in vitro molecular and functional characterization of the novel protein variant. Setting: Tertiary pediatric endocrine centers in Argentina and Switzerland. Patients: Five individuals (four 46,XY; one 46,XX) from four unrelated families presenting with disorders of sex development and adrenal dysfunction. Main Outcome Measures: Clinical phenotypes, hormonal profiles, and POR gene sequencing. In vitro analysis of recombinant POR measured flavin content, reductase activity, and support of steroidogenic and drug-metabolizing P450s. Results: All patients were homozygous for the c.262G>A (p.G88S) mutation. This FMN-binding domain variant caused protein instability with severe loss of FMN (Conclusions: The POR G88S mutation causes one of the most severe forms of PORD described, driven by dynamic protein instability and cofactor loss. It is a critical pharmacogenomic marker, and its recurrence in Argentina suggests a potential screening target.

Pubertal delay can be due to hypogonadotropic hypogonadism (HH), which may occur in association with anosmia or hyposmia and is known as Kallmann syndrome (OMIM #308700). Recently, hypogonadotropic hypogonadism has been suggested to overlap with Witteveen–Kolk syndrome (WITKOS, OMIM #613406) associated with 15q24 microdeletions encompassing SIN3A. Whether hypogonadotropic hypogonadism is due to haploinsufficiency of SIN3A or any of the other eight genes present in 15q24 is not known. We report the case of a female patient with delayed puberty associated with intellectual disability, behavior problems, dysmorphic facial features, and short stature, at the age of 14 years. Clinical, laboratory, and imaging assessments confirmed the diagnosis of Kallmann syndrome. Whole-exome sequencing identified a novel heterozygous frameshift variant, NM_001145358.2:c.3045_3046dup, NP_001138830.1:p.(Ile1016Argfs*6) in SIN3A, classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG/AMP) criteria. Reverse phenotyping led to the clinical diagnosis of WITKOS. No other variant was found in the 96 genes potentially related to hypogonadotropic hypogonadism. The analysis of the other contiguous seven genes to SIN3A in 15q24 did not reveal any clinically relevant variant. In conclusion, these findings point to SIN3A as the gene in 15q24 related to the reproductive phenotype in patients with overlapping WITKOS and Kallmann syndrome.

In patients with 46,XY disorders of sex development (DSDs), next-generation sequencing (NGS) has high diagnostic efficiency. One contribution to this diagnostic approach is the possibility of applying reverse phenotyping when a variant in a gene associated with multiple organ hits is found. Our aim is to report a case of a patient with 46,XY DSDs in whom the identification of a novel variant in MYRF led to the detection of a clinically inapparent congenital heart defect. A full-term newborn presented with ambiguous genitalia, as follows: a 2 cm phallus, penoscrotal hypospadias, partially fused labioscrotal folds, an anogenital distance of 1.2 cm, and non-palpable gonads. The karyotype was 46,XY, serum testosterone and AMH were low, whereas LH and FSH were high, leading to the diagnosis of dysgenetic DSD. Whole exome sequencing identified a novel, heterozygous, nonsense variant in MYRF, classified as pathogenic according to the ACMG criteria. MYRF encodes a membrane-bound transcriptional factor expressed in several tissues associated with OCUGS syndrome (ophthalmic, cardiac, and urogenital anomalies). In the patient, oriented clinical assessment ruled out ophthalmic defects, but ultrasonography confirmed meso/dextrocardia. We report a novel MYRF variant in a patient with 46,XY DSDs, allowing us to identify a clinically inapparent congenital heart defect by reverse phenotyping.

The diagnosis of male central (or hypogonadotropic) hypogonadism, typically based on low luteinizing hormone (LH) and testosterone levels, is challenging during childhood since both hormones are physiologically low from the sixth month until the onset of puberty. Conversely, follicle-stimulating hormone (FSH) and anti-Müllerian hormone (AMH), which show higher circulating levels during infancy and childhood, are not used as biomarkers for the condition. We report the case of a 7-year-old boy with a history of bilateral cryptorchidism who showed repeatedly low FSH and AMH serum levels during prepuberty. Unfortunately, the diagnosis could not be ascertained until he presented with delayed puberty at the age of 14 years. A gonadotropin-releasing hormone (GnRH) test showed impaired LH and FSH response. By then, his growth and bone mineralization were partially impaired. Gene panel sequencing identified a variant in exon 15 of FGFR1, affecting the tyrosine kinase domain of the receptor, involved in GnRH neuron migration and olfactory bulb morphogenesis. Testosterone replacement was started, which resulted in the development of secondary sexual characteristics and partial improvement of bone mineral density. This case illustrates the difficulty in making the diagnosis of central hypogonadism in boys during childhood based on classical criteria, and how serum FSH and AMH assessment may be helpful if it is suspected before the age of puberty, and confirm it using next-generation sequencing. The possibility of making an early diagnosis of central hypogonadism may be useful for a timely start of hormone replacement therapy, and to avoid delays that could affect growth and bone health as well as psychosocial adjustment.

In girls with Idiopathic Central Precocious Puberty (ICPP) concern has been raised by the potential impact of GnRH-analogues (GnRHa) treatment on body weight. We evaluated the effect of GnRHa on Body Mass Index (BMI) in girls with ICPP according to weight status at diagnosis. One hundred seventeen ICPP girls were divided according to pretreatment weight status in: normal weight (NW), overweight (OW) and obese (OB). BMI at one and two years of treatment was assessed. BMI-SDS of 60 patients who reached adult height (AH) was compared to that of 33 ICPP untreated girls. NW girls significantly increased their baseline BMI-SDS at 1 and 2 years of treatment. OW girls only had a significant increment at one year of treatment while OB girls showed no BMI-SDS change. Patients evaluated at AH (at least four years after GnRHa withdrawal) showed a significant decrease on BMI compared to baseline and a significantly lower BMI than the untreated group. In ICPP girls the BMI increase under GnRHa was inversely related to the pretreatment weight status. In the long term follow-up, no detrimental effect of GnRHa on body weight was observed. BMI-SDS was lower in treated than in untreated girls.