Loss-of-Function Variants in PPP1R12A: From Isolated Sex Reversal to Holoprosencephaly Spectrum and Urogenital Malformations.

In two independent ongoing next-generation sequencing projects for individuals with holoprosencephaly and individuals with disorders of sex development, and through international research collaboration, we identified twelve individuals with de novo loss-of-function (LoF) variants in protein phosphatase 1, regulatory subunit 12a (PPP1R12A), an important developmental gene involved in cell migration, adhesion, and morphogenesis. This gene has not been previously reported in association with human disease, and it has intolerance to LoF as illustrated by a very low observed-to-expected ratio of LoF variants in gnomAD. Of the twelve individuals, midline brain malformations were found in five, urogenital anomalies in nine, and a combination of both phenotypes in two. Other congenital anomalies identified included omphalocele, jejunal, and ileal atresia with aberrant mesenteric blood supply, and syndactyly. Six individuals had stop gain variants, five had a deletion or duplication resulting in a frameshift, and one had a canonical splice acceptor site loss. Murine and human in situ hybridization and immunostaining revealed PPP1R12A expression in the prosencephalic neural folds and protein localization in the lower urinary tract at critical periods for forebrain division and urogenital development. Based on these clinical and molecular findings, we propose the association of PPP1R12A pathogenic variants with a congenital malformations syndrome affecting the embryogenesis of the brain and genitourinary systems and including disorders of sex development.

Phase 3 Trial of a Small-volume Subcutaneous 6-Month Duration Leuprolide Acetate Treatment for Central Precocious Puberty.

CONTEXT: Gonadotropin-releasing hormone agonists (GnRHas) are standard of care for central precocious puberty (CPP). A 6-month subcutaneous injection has recently been approved by the Food and Drug Administration. OBJECTIVE: Determine efficacy, pharmacokinetics, and safety of 6-month 45-mg subcutaneous leuprolide acetate for CPP. DESIGN: Phase 3 multicenter, open-label, single-arm study. SETTING: 25 sites in 6 countries. SUBJECTS: 64 GnRHa-naïve children with CPP (age: 7.5 ± 0.1 years) received study drug: 59 completed the study. INTERVENTION(S): 2 doses of 45-mg subcutaneous leuprolide acetate (0.375 mL) at 0 and 24 weeks; children were followed for 48 weeks. MAIN OUTCOME MEASURE(S): Percentage of children with serum luteinizing hormone (LH) <4 IU/L 30 minutes following GnRHa stimulation at week 24. RESULTS: 54/62 (87%) children achieved poststimulation LH <4 IU/L at week 24; 49/56 (88%) girls and 1/2 boys maintained peak LH <4 IU/L at week 48. Mean growth velocity decreased from 8.9 cm/year at week 4 to 6.0 cm/year at week 48. Mean bone age was advanced 3.0 years beyond chronological age at screening and 2.7 years at week 48. Breast pubertal stage regressed or was stable in 97% of girls and external genitalia development regressed in both boys. Adverse events were mild and did not cause treatment discontinuation. CONCLUSIONS: A small volume of 45-mg subcutaneous leuprolide acetate administered at a 6-month interval effectively suppressed pubertal hormones and stopped or caused regression of pubertal progression. This long-acting GnRHa preparation of leuprolide acetate is a new, effective, and well-tolerated therapy for children with CPP.

Efficacy and safety of histrelin subdermal implant in children with central precocious puberty: a multicenter trial.

CONTEXT: GnRH analog (GnRHa) therapy for central precocious puberty (CPP) typically involves im injections. The histrelin implant is a new treatment that provides a continuous slow release of the GnRHa histrelin. OBJECTIVE: The objective of the study was to investigate the safety and efficacy of the subdermal histrelin implant for the treatment of CPP in treatment naive and previously treated children. DESIGN: This was a phase III, open-label, prospective study of 1-yr duration. SETTING: The study was conducted at nine U.S. medical centers. PATIENTS: Girls ages 2-8 yr (naive) or 2-10 yr (previously treated) and boys 2-9 yr (naive) or 2-11 yr (previously treated) with clinical evidence of CPP and a pretreatment pubertal response to leuprolide stimulation were eligible. INTERVENTION: A 50-mg histrelin implant was inserted sc in the inner upper arm. MAIN OUTCOME MEASURES: Peak LH after GnRHa stimulation testing and estradiol (girls) and testosterone (boys) were the main outcome measures. RESULTS: Thirty-six subjects (20 naive) were enrolled. By 1 month, peak LH fell from 28.2 +/- 19.97 (naive) to 0.8 +/- 0.39 mIU/ml (P < 0.0001) and from 2.1 +/- 2.15 (previously treated) to 0.5 +/- 0.32 mIU/ml (P < 0.0056). Estradiol suppressed from 24.5 +/- 22.27 (naive) to 5.9 +/- 2.37 pg/ml (P = 0.0016) and remained suppressed in previously treated subjects, as did testosterone. Suppression was maintained throughout the study. No significant adverse events occurred. CONCLUSIONS: The subdermal histrelin implant achieves and maintains excellent suppression of peak LH and sex steroid levels for 1 yr in children with CPP. The treatment is well tolerated. Long-term studies are needed to confirm these results.

Remission of congenital diabetes insipidus after eight years.

Septo-optic dysplasia (SOD) (De Morsier's syndrome) is a complex developmental disorder marked by variable and often incomplete formation of cranial midline structures, resulting in absence of the septum pellucidum, optic nerve hypoplasia, and hypothalamic-pituitary dysfunction. We describe a patient with SOD who manifested symptoms in the early neonatal period with severe deficiencies of multiple pituitary hormones including anti-diuretic hormone (ADH). Her congenital diabetic insipidus (DI), consequence of an anatomic defect, can be argued to be of the most severe type. Our patient resolved her severe DI 8 years after her initial presentation, suddenly requiring no further medical treatment for DI following longstanding pharmacological replacement of ADH. This is the first report of a patient with SOD with spontaneous resolution of congenital DI.

Adrenocorticotropin-secreting pancreatoblastoma.

We present a 3-year-old child with Cushing's syndrome due to an ACTH-secreting metastatic pancreatoblastoma. This malignancy is a rare cause of Cushing's syndrome, particularly at pediatric age. We describe her course including the use of ketoconazole to alleviate hypercortisolemia.

A Randomized Safety and Efficacy Study of Somavaratan (VRS-317), a Long-Acting rhGH, in Pediatric Growth Hormone Deficiency.

CONTEXT: Somavaratan (VRS-317) is a long-acting form of recombinant human GH under development for children and adults with GH deficiency (GHD). OBJECTIVES: To determine the optimal somavaratan dose regimen to normalize IGF-1 in pediatric GHD and to evaluate safety and efficacy of somavaratan over 6 months. DESIGN: Open-label, multicenter, single ascending dose study followed by 6-month randomized comparison of 3 dosing regimens. SETTING: Twenty-five United States pediatric endocrinology centers. PATIENTS: Naive-to-treatment, prepubertal children with GHD (n = 68). INTERVENTION(S): Patients received single sc doses of somavaratan (0.8, 1.2, 1.8, 2.7, 4.0, or 6.0 mg/kg) during the 30-day dose-finding phase, then were randomized to somavaratan 1.15 mg/kg weekly, 2.5 mg/kg twice monthly, or 5.0 mg/kg monthly for 6 months. MAIN OUTCOME MEASURES: Safety, pharmacokinetics, pharmacodynamics, 6-month height velocity (HV). RESULTS: Somavaratan pharmacokinetics was linearly proportional to dose; dose-dependent increases in the magnitude and duration of IGF-1 responses enabled weekly, twice-monthly or monthly dosing. A single dose of somavaratan sustained IGF-1 responses for up to 1 month. No somavaratan or IGF-1 accumulation occurred with repeat dosing. Mean annualized HVs for somavaratan administered monthly, twice monthly, or weekly (7.86 ± 2.5, 8.61 ± 2.7, and 7.58 ± 2.5 cm/y, respectively) were similar between groups. Adverse events were mostly mild and transient. CONCLUSIONS: Somavaratan demonstrated clinically meaningful improvements in HV and IGF-1 in prepubertal children with GHD, with no significant differences between monthly, twice-monthly, or weekly dosing.

Long-Term Continuous Suppression With Once-Yearly Histrelin Subcutaneous Implants for the Treatment of Central Precocious Puberty: A Final Report of a Phase 3 Multicenter Trial.

CONTEXT AND OBJECTIVE: The histrelin implant has proven to be an effective method of delivering GnRH analog (GnRHa) therapy to children with central precocious puberty (CPP), yet there are limited data available regarding hormonal suppression and auxological changes during an extended course of therapy. DESIGN: This was a phase 3, prospective, open-label study. SETTING AND PARTICIPANTS: Thirty-six children with CPP who participated in a phase 3, open-label study and required further GnRHa therapy were eligible to continue treatment receiving a new implant upon removal of the prior 12-month histrelin implant during a long-term extension phase. OUTCOME MEASURES: Hormone levels and auxologic parameters were measured periodically for up to 6 years of treatment and up to 1 year of posttreatment follow-up. RESULTS: Hormonal suppression was maintained throughout the study in patients who had prior GnRHa therapy (n = 16) and in treatment-naive patients (n = 20). Bone age to chronological age ratio decreased from 1.417 (n = 20) at baseline to 1.18 (n = 8) at 48 months in treatment-naive children (P < .01). Predicted adult height in girls increased from 151.9 cm at baseline to 166.5 cm at month 60 (n = 6; P < .05), with a 10.7-cm height gain observed among treatment-naive children (n = 5). No adverse effect on growth or recovery of the hypothalamic-pituitary-gonadal axis was observed with hormonal suppression. The histrelin implant was generally well tolerated during long-term therapy. CONCLUSIONS: Long-term histrelin implant therapy provided sustained gonadotropin suppression safely and effectively and improved predicted adult height in children with CPP.

Micropenis with testicular regression, low LH levels, and poor androgen and HCG responses: a distinct syndrome?

We report three boys, including two brothers, with micropenis and poor phallic growth in response to both exogenous human chorionic gonadotropin (HCG) and testosterone therapy in the newborn period. They exhibited low neonatal testosterone levels that failed to respond to HCG stimulation. These boys displayed a unique gonadotropin profile with reduced luteinizing hormone levels and elevated follicle-stimulating hormone levels. They had small, cryptorchid testes with subsequent testicular regression and atrophy. Moreover, all three boys have developed microcephaly and mild learning delays. We review the hormonal profiles and phenotypes of known causes of micropenis, and compare them to the features of our three patients. Although individuals with similar features may have been described in past series of males with micropenis, the data presented previously were insufficient for classification; thus, we propose that our patients may represent a distinct, not previously recognized, syndrome with either X-linked recessive inheritance or autosomal recessive inheritance with male sex limitation.

Tumor localization and biochemical response to cure in tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) is a rare disorder of phosphate wasting due to fibroblast growth factor-23 (FGF23)-secreting tumors that are often difficult to locate. We present a systematic approach to tumor localization and postoperative biochemical changes in 31 subjects with TIO. All had failed either initial localization, or relocalization (in case of recurrence or metastases) at outside institutions. Functional imaging with ¹¹¹Indium-octreotide with single photon emission computed tomography (octreo-SPECT or SPECT/CT), and ¹8fluorodeoxyglucose positron emission tomography/CT (FDG-PET/CT) were performed, followed by anatomic imaging (CT, MRI). Selective venous sampling (VS) was performed when multiple suspicious lesions were identified or high surgical risk was a concern. Tumors were localized in 20 of 31 subjects (64.5%). Nineteen of 20 subjects underwent octreo-SPECT imaging, and 16 of 20 FDG-PET/CT imaging. Eighteen of 19 (95%) were positive on octreo-SPECT, and 14 of 16 (88%) on FDG-PET/CT. Twelve of 20 subjects underwent VS; 10 of 12 (83%) were positive. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: sensitivity = 0.95, specificity = 0.64, PPV = 0.82, and NPV = 0.88 for octreo-SPECT; sensitivity = 0.88, specificity = 0.36, PPV = 0.62, and NPV = 0.50 for FDG-PET/CT. Fifteen subjects had their tumor resected at our institution, and were disease-free at last follow-up. Serum phosphorus returned to normal in all subjects within 1 to 5 days. In 10 subjects who were followed for at least 7 days postoperatively, intact FGF23 (iFGF23) decreased to near undetectable within hours and returned to the normal range within 5 days. C-terminal FGF23 (cFGF23) decreased immediately but remained elevated, yielding a markedly elevated cFGF23/iFGF23 ratio. Serum 1,25-dihydroxyvitamin D3 (1,25D) rose and exceeded the normal range. In this systematic approach to tumor localization in TIO, octreo-SPECT was more sensitive and specific, but in many cases FDG-PET/CT was complementary. VS can discriminate between multiple suspicious lesions and increase certainty prior to surgery. Sustained elevations in cFGF23 and 1,25D were observed, suggesting novel regulation of FGF23 processing and 1,25D generation.

Inherited germline TP53 mutation encodes a protein with an aberrant C-terminal motif in a case of pediatric adrenocortical tumor.

Childhood adrenocortical tumor (ACT), a very rare malignancy, has an annual worldwide incidence of about 0.3 per million children younger than 15 years. The association between inherited germline mutations of the TP53 gene and an increased predisposition to ACT was described in the context of the Li-Fraumeni syndrome. In fact, about two-thirds of children with ACT have a TP53 mutation. However, less than 10% of pediatric ACT cases occur in Li-Fraumeni syndrome, suggesting that inherited low-penetrance TP53 mutations play an important role in pediatric adrenal cortex tumorigenesis. We identified a novel inherited germline TP53 mutation affecting the acceptor splice site at intron 10 in a child with an ACT and no family history of cancer. The lack of family history of cancer and previous information about the carcinogenic potential of the mutation led us to further characterize it. Bioinformatics analysis showed that the non-natural and highly hydrophobic C-terminal segment of the frame-shifted mutant p53 protein may disrupt its tumor suppressor function by causing misfolding and aggregation. Our findings highlight the clinical and genetic counseling dilemmas that arise when an inherited TP53 mutation is found in a child with ACT without relatives with Li-Fraumeni-component tumors.

Results of a second year of therapy with the 12-month histrelin implant for the treatment of central precocious puberty.

Background. Gonadotropin releasing hormone analogs (GnRHas) are standard of care for central precocious puberty (CPP). The histrelin subcutaneous implant is safe and effective in the treatment of CPP for one year. Objective. The study evaluates a second year of therapy in children with CPP who received a new implant after one year of treatment. Methods. A prospective one-year study following an initial 12-month treatment period was conducted. Results. Thirty-one patients (29 girls) aged 7.7 +/- 1.5 years received a second implant. Eighteen were naïve to GnRHa therapy at first implantation. Peak LH declined from 0.92 +/- 0.58 mIU/mL at 12 months to 0.51 +/- 0.33 mIU/mL at 24 months (P < .0001) in naïve subjects, and from 0.74 +/- 0.50 mIU/mL at 12 months to 0.45 +/- 0.35 mIU/mL at 24 months (P = .0081) in previously treated subjects. Predicted adult height increased by 5.1 cm at 24 months (P = .0001). Minor implant site reactions occurred in 61%, while minor difficulties with explantation occurred in 32.2% of subjects. Conclusion. The histrelin implant demonstrates profound hypothalamic-pituitary-gonadal axis suppression when a new implant is placed for a second year of treatment. Prospective follow-up of this therapeutic modality for the treatment of CPP is needed.

Onset of acquired autoimmune hypothyroidism in infancy: a presentation of delayed gross-motor development and rhabdomyolysis.

We report the case of a 23-month-old girl who presented with poor growth and delayed attainment of gross-motor milestones. Elevated creatine phosphokinase (CPK) indicated rhabdomyolysis, ultimately attributed to severe, acquired autoimmune hypothyroidism. Growth data and bone-age suggest the onset of hypothyroidism occurred at or before 12 months of age. Acquired hypothyroidism is rare before age 3 years, and rhabdomyolysis due to hypothyroidism has not previously been reported as a cause of delayed gross-motor development in toddlerhood. Despite the early onset of hypothyroidism, cognitive function appeared to be unaffected. Adequate thyroid hormone replacement quickly normalized the CPK in our patient, and gross motor development rapidly improved. Although rare, rhabdomyolysis secondary to hypothyroidism should be in the differential diagnosis of delayed gross-motor development in infancy and toddlerhood.

A familial missense mutation in the hinge region of DAX1 associated with late-onset AHC in a prepubertal female.

Mutations in the DAX1 (Dosage-sensitive sex reversal-Adrenal hypoplasia congenita (AHC) critical region on the X chromosome gene 1; NR0B1) cause X-linked AHC, a disease characterized by primary adrenal failure in infancy and hypogonadotropic hypogonadism. All known missense mutations impair DAX1 repression of steroidogenic factor 1 (SF1) transactivation and have been localized to the putative ligand binding domain. Here, an asymptomatic father and his late-onset AHC daughter were both shown to share a novel DAX1 mutation (C200W), the first missense mutation identified in the hinge region of DAX1. Using immunohistochemistry we demonstrate that the sub-cellular localization of the C200W mutant DAX1 protein is shifted from the nucleus to the cytoplasm. The disturbed localization of the C200W mutant in transfected cells correlates with impaired transcriptional repression activity. The import defect is relatively mild, retaining 80% of wild-type activity, which may explain the unusually mild phenotype. Import of DAX1 into the nucleus involves a direct interaction with SF1. In vitro assays demonstrate that the C200W mutant retains the ability to functionally interact with SF1, which suggests that SF1-independent interactions of DAX1 could be responsible for the import defect.

Agenesis of the mesencephalon and metencephalon with cerebellar hypoplasia: putative mutation in the EN2 gene--report of 2 cases in early infancy.

Congenital absence of the midbrain and upper pons is a rare human malformation. We describe two unrelated infants with this anomaly and cerebellar hypoplasia who were born at term but died in early infancy from lack of central respiratory drive. MRI in both cases disclosed the lesions during life. Neuropathological examination, performed in one, included immunocytochemical studies of NeuN, synaptophysin, vimentin, and glial fibrillary acidic protein (GFAP). Autopsy revealed a thin midline cord passing through the clivus, in place of the midbrain; it corresponded to hypoplastic and fused corticospinal tracts with ectopic neural tissue in the surrounding leptomeninges. Some ectopia were immunoreactive for synaptophysin and NeuN and others were nonreactive. The neural surfaces facing the subarachnoid fluid-filled space left by the absent midbrain and upper pons were lined by an abnormal villous ependyma. The architecture of the cerebellar cortex was imperfect but generally normal, and Bergmann glial cells had normal radial processes shown by vimentin and GFAP. Structures of the telencephalon, diencephalon, lower brainstem, and spinal cord were generally well formed, but inferior olivary and dentate nuclei were rudimentary and the spinal central canal was dilated at lumbar levels. The cerebral cortex was normally laminated, but pyramidal neurons of layer 5 were sparse in the frontal lobes. The hippocampus, olfactory system, and corpus callosum were formed. An ectopic lingual thyroid was found and had been associated with hypothyroidism during life. A murine model resembling this dysgenesis is demonstrated by homozygous mutations of the organizer genes Wnt1 or En1, also resulting in cerebellar aplasia, and En2, associated with cerebellar hypoplasia. These genes are essential to the formation of the mesencephalic neuromere and rhombomere 1 (metencephalon or upper pons and cerebellum). Pax8 has binding sites in the promoter for En2 and is essential for thyroid development. We speculate that in the human, the failure to form a mesencephalon and metencephalon, with cerebellar hypoplasia, results from a mutation or deletion in the EN2 (Engrailed-2) gene.