Severe aldosterone synthase deficiency in a nine-day old Lebanese boy: the importance of functional studies to establish pathogenicity of seemingly benign variants in CYP11B2.

Introduction Aldosterone synthase deficiency is a rare autosomal recessive disease characterized by vomiting, dehydration, salt wasting, life-threatening hyperkalemia in infancy, followed by failure to thrive. It results from pathogenic variants in CYP11B2. Case Presentation A boy, born in Montreal to Lebanese parents who are first cousins, was referred at nine days of life for severe dehydration. A diagnosis of primary adrenal insufficiency was made, and treatment was started with fludrocortisone and hydrocortisone. Exome sequencing revealed a homozygous variant p.(Asn201Asp)(N201D). In silico, this variant was considered benign, but in vitro functional expression studies established it caused the severe aldosterone deficiency. It ended the diagnostic odyssey and allowed to safely stop hydrocortisone replacement. Conclusion If a gene variant co-segregates with a phenotype, in vitro functional studies are required even if in silico studies are negative.

Whole-Exome Sequencing in Congenital Hypothyroidism Due to Thyroid Dysgenesis.

Context: Congenital hypothyroidism due to thyroid dysgenesis (CHTD) is a predominantly sporadic and nonsyndromic (NS) condition of unknown etiology. NS-CHTD shows a 40-fold increase in relative risk among first-degree relatives (1 in 100 compared with a birth prevalence of 1 in 4000 in the general population), but a discordance rate between monozygotic (MZ) twins of 92%. This suggests a two-hit mechanism, combining a genetic predisposition (incomplete penetrance of inherited variants) with postzygotic events (accounting for MZ twin discordance). Objective: To evaluate whether whole-exome sequencing (WES) allows to identify new predisposing genes in NS-CHTD. Methods: We performed a case-control study by comparing the whole exome of 36 nonconsanguineous cases of NS-CHTD (33 with lingual thyroid ectopy and 3 with athyreosis, based on technetium pertechnetate scintigraphy at diagnosis) with that of 301 unaffected controls to assess for enrichment in rare protein-altering variants. We performed an unbiased approach using a gene-based burden with a false discovery rate correction. Moreover, we identified all rare pathogenic and likely pathogenic variants, based on in silico prediction tools, in 27 genes previously associated with congenital hypothyroidism (CH) (thyroid dysgenesis [TD] and dyshormonogenesis). Results: After correction for multiple testing, no enrichment in rare protein-altering variants was observed in NS-CHTD. Pathogenic or likely pathogenic variants (21 variants in 12 CH genes) were identified in 42% of cases. Eight percent of cases had variants in more than one gene (oligogenic group); these were not more severely affected than monogenic cases. Moreover, cases with protein-altering variants in dyshormonogenesis-related genes were not more severely affected than those without. Conclusions: No new predisposing genes were identified following an unbiased analysis of WES data in a well-characterized NS-CHTD cohort. Nonetheless, the discovery rate of rare pathogenic or likely pathogenic variants was 42%. Eight percent of the cases harbored multiple variants in genes associated with TD or dyshormonogenesis, but these variants did not explain the variability of hypothyroidism observed in dysgenesis. WES did not identify a genetic cause in NS-CHTD cases, confirming the complex etiology of this disease. Additional studies in larger cohorts and/or novel discovery approaches are required.

Severe Congenital Hypothyroidism Due to a Novel Deep Intronic Mutation in the TSH Receptor Gene Causing Intron Retention.

In 3 Somalian siblings with severe nongoitrous congenital hypothyroidism, exome sequencing identified a variant in TSHR predicted to be benign in isoform 3 but leading to an intronic mutation in isoform 1 (NM_00369:c.692 + 130C>A), which is the isoform expressed in the thyroid. This mutation creates a pseudoexon that results in a protein that, if transcribed, would lack the transmembrane domain, thereby hampering its expression at the cell surface. Our findings illustrate that the interpretation of exome analysis requires knowledge of the relevant isoform expression and of the biology of the disease. This is the first description of a deep intronic mutation creating a pseudoexon and inactivating the thyroid stimulating hormone (TSH) receptor.

Wide Spectrum of DUOX2 Deficiency: From Life-Threatening Compressive Goiter in Infancy to Lifelong Euthyroidism.

Six patients are described with bi-allelic DUOX2 variants and widely variable phenotypes. Patient 1 is an infant with a compressive hypothyroid goiter causing respiratory distress, which was promptly alleviated by levothyroxine (LT4). He was a compound heterozygote for DUOX2 variants, including a novel deletion of 540 base pairs. Patients 2 and 3 are siblings with the same compound heterozygous mutations of DUOX2, yet one had overt hypothyroidism at 14 months and the other lifelong euthyroidism. Patient 4 is a compound heterozygote individual and has mild persistent congenital hypothyroidism; his sister (patient 5) only had a borderline thyrotropin elevation at newborn screening, consistent with homozygous DUOX2 variants with a mild impact on enzyme activity. Their euthyroid mother (patient 6) is a compound heterozygote for the same DUOX2 mutations as her son. Targeted exome sequencing did not reveal any relevant modifiers. It is concluded that (i) prompt LT4 replacement in infants with respiratory distress due to a hypothyroid goiter makes surgery unnecessary; and (ii) the clinical expression of DUOX2 deficiency varies widely between individuals and over time, justifying periodic reevaluation of the need for LT4 replacement.

Pediatric thyroidectomy: Favorable outcomes can be achieved by a multidisciplinary team of pediatric providers.

AIM OF THE STUDY: Recent publications suggest pediatric surgeons may not be well suited to perform thyroid surgeries unless considered high volume. We sought to assess the outcome of thyroidectomies performed by pediatric surgeons in an academic setting. METHODS: We reviewed charts of patients younger than 18 years who underwent thyroid surgeries at a free standing children's hospital between April 2006 and October 2015. MAIN RESULTS: The analysis included 118 surgeries in 98 patients (mean age 11.8 years). Most surgeries were performed by a single pediatric surgeon (average 10 thyroidectomies per year). The commonest indication for resection was thyroid nodule (64%). 80% of patients had a single surgery; the remainder had two, including 13 completion hemithyroidectomies. Cancer was found in 37% of specimens, with papillary subtype being most common (72%). Seven patients had locoregional metastases and one had pulmonary metastases. Among the 17 malignant cases that had a second intervention, 6 had malignancy in the resected specimen. There were no deaths in the follow up period (mean 2.7 years). Two patients had permanent hypocalcemia, and three had persistent unilateral recurrent laryngeal nerve injuries causing dysphonia for a total permanent complication rate of 4.2%. CONCLUSIONS: We conclude that pediatric thyroidectomy is a safe procedure when performed by pediatric surgeons. Our rate of complications is comparable to those reported in the literature. Our data highlight the need for a vigilant and multidisciplinary approach for children with thyroid pathology. TYPE OF STUDY: Treatment study. LEVEL OF EVIDENCE: IV.

Prophylactic thyroidectomies in MEN2 syndrome: Management and outcomes.

AIM OF THE STUDY: The aim of the study was to evaluate the outcomes of prophylactic thyroidectomies performed in an academic setting in the context of multiple endocrine neoplasia type 2 (MEN2) syndrome. METHODS: A chart review of patients <18years old who underwent prophylactic thyroidectomy for a MEN2 syndrome at a children's hospital between 2006 and 2015 was performed. MAIN RESULTS: The study included 21 patients (57% female) with a mean age of 6.2±2.5years. All patients were asymptomatic at first evaluation. Nineteen had MEN2A syndrome with RET proto-oncogene mutations identified. The remaining two were RET-negative with familial medullary thyroid cancer (FMTC). One patient had a concomitant Hirschsprung disease. Of the 11 patients who had RET proto-oncogene mutations ranked as Moderate Risk for medullary thyroid cancer (MTC) (American Thyroid Association), one had a microcarcinoma on the resected specimen, and the others had C-Cell Hyperplasia. Among the 8 patients who had RET proto-oncogene mutations ranked as High Risk level for MTC, all had microcarcinoma. Of the nine patients with microcarcinoma, three underwent surgery after 5years of age. No microcarcinoma exceeded 6mm. There were no permanent complications. Six patients experienced transient hypocalcemia, of which only one was symptomatic. No patients had lymph node involvement, and no recurrence was noted during the follow-up period. CONCLUSIONS: Of 21 children with familial thyroid cancer syndrome who underwent a prophylactic thyroidectomy, nine had microcarcinoma. This study highlights the need for a complete familial history, including FMTC history and mandatory preventive surgical approach. LEVEL OF EVIDENCE: III.

Disorders of thyroid morphogenesis.

Developmental anomalies of the thyroid gland, defined as thyroid dysgenesis, underlie the majority of cases of congenital hypothyroidism. Thyroid dysgenesis is predominantly a sporadic disorder although a reported familial enrichment, variation of incidence by ethnicity and the monogenic defects associated mainly with athyreosis or orthotopic thyroid hypoplasia, suggest a genetic contribution. Of note, the most common developmental anomaly, thyroid ectopy, remains unexplained. Ectopy may result from multiple genetic or epigenetic variants in the germline and/or at the somatic level. This review provides a brief overview of the monogenic defects in candidate genes that have been identified so far and of the syndromes which are known to be associated with thyroid dysgenesis.

Continuous Subcutaneous Insulin Infusion in Children: A Pilot Study Validating a Protocol to Avoid Hypoglycemia at Initiation.

BACKGROUND: The occurrence of hypoglycemia and hyperglycemia during the first days after transition to continuous subcutaneous insulin infusion (CSII) in patients with type 1 diabetes has not been systematically studied in children. The aim of this prospective study was to demonstrate that the protocol applied in our diabetes clinic is safe at CSII initiation in children. METHODS: We assessed 22 pediatric patients with type 1 diabetes, using continuous glucose monitoring (CGM) before and after CSII initiation (±3 days). RESULTS: After CSII initiation, there was no difference in the rates of hypoglycemic events expressed as relative rates (RRs) per person-reading (RR = 0.85, p = 0.52, 95% CI 0.52-1.39), as well as in the number of prolonged hypoglycemic events (>1 h) per day (RR = 1.12, p = 0.56, 95% CI 0.75-1.68). We observed only a trend toward prolonged episodes of hyperglycemia after pump initiation (RR = 1.52, p = 0.06, 95% CI 0.97-2.35). CONCLUSION: Our study is the first to assess, through CGM and in a prospective way, the impact of a CSII initiation protocol on glycemic values. Our protocol provides a safe model to avoid hypoglycemia at CSII initiation in children. CLINICAL TRIAL REGISTRATION: www.ClinicalTrials.gov, identifier NCT01840358.

From Bioinactive ACTH to ACTH Antagonist: The Clinical Perspective.

The adrenocorticotropic hormone (ACTH) is a pituitary hormone derived from a larger peptide, the proopiomelanocortin (POMC), as are the MSHs (α-MSH, ß-MSH, and γ-MSH) and the ß-LPH-related polypeptides (Figure 1A). ACTH drives adrenal steroidogenesis and growth of the adrenal gland. ACTH is a 39 amino acid polypeptide that binds and activates its cognate receptor [melanocortin receptor 2 (MC2R)] through the two regions H6F7R8W9 and K15K16R17R18P19. Most POMC-derived polypeptides contain the H6F7R8W9 sequence that is conserved through evolution. This explains the difficulties in developing selective agonists or antagonists to the MCRs. In this review, we will discuss the clinical aspects of the role of ACTH in physiology and disease, and potential clinical use of selective ACTH antagonists.

Demonstration of Autosomal Monoallelic Expression in Thyroid Tissue Assessed by Whole-Exome and Bulk RNA Sequencing.

BACKGROUND: Congenital hypothyroidism due to thyroid dysgenesis (CHTD) is a disorder with a prevalence of 1/4000 live births, the cause of which remains unknown. The most common diagnostic category is thyroid ectopy, which occurs in up to 80% of CHTD cases. CHTD is predominantly not inherited and has a high discordance rate (>92%) between monozygotic (MZ) twins. The sporadic nature of CHTD might be explained by somatic events such as autosomal monoallelic expression (AME), given that genes expressed in a monoallelic way are more vulnerable to otherwise benign monoallelict genetic or epigenetic mutations. OBJECTIVE: The aim of this study was to search for complete (90%) AME in normal and dysgenetic thyroid tissues. METHODS: Aggregated analysis of whole-exome and bulk RNA sequencing was performed on two ectopic thyroids, four normal thyroids, and the human thyroid cell line Nthy-ori. RESULTS: A median of 5062 (range 2081-5270) genes per sample showed sufficient numbers of heterozygous single nucleotide polymorphisms to be informative. The median monoallelic expression represented 22 (range 16-32) of the informative genes for each thyroid sample. Examples of genes displaying AME are FCGBP, ZNF331, USP10, BCLAF1, and some HLA genes; these genes are involved in epithelial-mesenchymal transition, cell migration, cancer, and immunity. CONCLUSIONS: AME may account for the high discordance rate observed between MZ twins and for the sporadic nature of CHTD. These findings also have implications for other pathologies, including cancers and autoimmune disorders of the thyroid.

Influence of Adiposity, Physical Activity, Fitness, and Screen Time on Insulin Dynamics Over 2 Years in Children.

IMPORTANCE: Despite extensive evidence showing that lifestyle habits play a critical role in preventing or delaying the onset of type 2 diabetes in adults, little is known regarding the impact of lifestyle habits on type 2 diabetes risk in childhood. OBJECTIVE: To assess whether adiposity, fitness, moderate-to-vigorous physical activity, and screen time predict insulin sensitivity or insulin secretion during a 2-year period in children with a family history of obesity. DESIGN, SETTING, AND PARTICIPANTS: This is a prospective longitudinal cohort study of 630 children, having at least 1 obese parent, recruited from schools in Quebec, Canada, between July 2005 and December 2008 in the Quebec Adipose and Lifestyle Investigation in Youth (QUALITY) cohort. Children were assessed at baseline (ages 8-10 years) and 2 years later. Fitness was measured by peak oxygen consumption, percentage of body fat (adiposity) by dual-energy x-ray absorptiometry, moderate-to-vigorous physical activity using accelerometry, and screen time by average daily hours of self-reported television, video game, or computer use. Regression models were adjusted for age, sex, season, and pubertal stage. The current analysis was completed in October 2015. MAIN OUTCOMES AND MEASURES: Insulin sensitivity was measured by the homeostatic model assessment of insulin resistance and an oral glucose tolerance test-based index (Matsuda insulin sensitivity index). Insulin secretion was measured using the area under the curve of insulin to glucose during the first 30 minutes of the oral glucose tolerance test and using the area under the curve of insulin to glucose over 2 hours. RESULTS: Of 630 children evaluated at baseline (mean [SD] age, 9.6 [0.9] years; 54.4% male; 56.2% normal weight, 19.2% overweight, and 22.7% obese), 564 were evaluated at 2-year follow-up. Adiposity and changes in adiposity were the central predictors of insulin dynamics over time. Every additional 1% of body fat at ages 8 to 10 years decreased insulin sensitivity by 2.9% (95% CI, -3.3% to -2.5%; P < .001) and led to a 0.5% (95% CI, 0.09% to 0.8%; P = .02) increased requirement in the area under the curve of insulin to glucose during the first 30 minutes of the oral glucose tolerance test 2 years later. Higher levels of moderate-to-vigorous physical activity and lower screen time appear to be beneficial to insulin sensitivity in part through their effect on adiposity levels. CONCLUSIONS AND RELEVANCE: Adiposity plays a determining role in cardiometabolic health at a young age. Public health strategies that promote healthy body weight, notably physical activity, need to target school-aged and possibly younger children.

Similar age-dependent levothyroxine requirements of schoolchildren with congenital or acquired hypothyroidism.

UNLABELLED: A recent study in children suggested that levothyroxine requirements are higher in congenital than in acquired hypothyroidism but did not match for severity of disease. Here, we studied only children with congenital or acquired hypothyroidism who had an undetectable fT4 at diagnosis. There were eight girls with congenital hypothyroidism due to athyreosis and eight girls with acquired hypothyroidism due to autoimmune thyroid disease. The median levothyroxine dose received at the most recent visit when serum TSH was <5.0 mU/L (at a median age of 7.86 and 14.29 years, respectively) was 3.2 mcg/kg/day in the former and 2.4 mcg/kg/day in the latter (N.S.). Combining both groups, the levothyroxine requirement decreased by 0.5 mcg/kg/day for every 4-year period. CONCLUSION: When strictly matched for severity of disease, levothyroxine requirements are similar in school-age children with congenital or acquired hypothyroidism and decrease with age. Thus, in congenital hypothyroidism treated early with high-dose levothyroxine, pituitary resistance to thyroxine feedback does not appear to be present at school age. WHAT IS KNOWN: • Pediatric studies unmatched for severity have suggested that levothyroxine requirements are higher in congenital than in acquired hypothyroidism. What is new: • When strictly matched for severity, levothyroxine requirements are similar in children with congenital or acquired hypothyroidism and decrease with age.

Early-Onset Central Diabetes Insipidus due to Compound Heterozygosity for AVP Mutations.

BACKGROUND: Genetic cases of isolated central diabetes insipidus are rare, are mostly due to dominant AVP mutations and have a delayed onset of symptoms. Only 3 consanguineous pedigrees with a recessive form have been published. CASE REPORT: A boy with a negative family history presented polyuria and failure to thrive in the first months of life and was diagnosed with central diabetes insipidus. Magnetic resonance imaging showed a normal posterior pituitary signal. A molecular genetic analysis of the AVP gene showed that he had inherited a previously reported mutation from his Lebanese father and a novel A>G transition in the splice acceptor site of intron 1 (IVS1-2A>G) from his French-Canadian mother. Replacement therapy resulted in the immediate disappearance of symptoms and in weight gain. CONCLUSIONS: The early polyuria in recessive central diabetes insipidus contrasts with the delayed presentation in patients with monoallelic AVP mutations. This diagnosis needs to be considered in infants with very early onset of polyuria-polydipsia and no brain malformation, even if there is no consanguinity and regardless of whether the posterior pituitary is visible or not on imaging. In addition to informing family counseling, making a molecular diagnosis eliminates the need for repeated imaging studies.

Interpreting Minor Variations in Thyroid Function or Echostructure: Treating Patients, Not Numbers or Images.

Overt thyroid dysfunction is documented by serum thyrotropin or T4 concentrations are often ordered for nonspecific complaints and will by definition fall outside of the 95% reference range 5% of the time. In addition, most laboratories quote adult ranges, which are not necessarily applicable to young children, and regression toward the mean is common, justifying that the test be repeated before embarking on treatment. On the other hand, neck ultrasounds are frequently performed for diffuse goiter or non-thyroid conditions. Yet, an ultrasound is not required to make a diagnosis of Hashimoto thyroiditis and small cysts and nodules discovered incidentally often lead to unjustified concerns about neoplasia.

Conserved Telomere Length in Human Ectopic Thyroids: An Argument Against Premature Differentiation Causing Arrested Migration.

BACKGROUND: In humans, the cause of arrested migration of the median thyroid anlage resulting in an ectopic sublingual gland is unknown. These ectopic glands have a normal follicular architecture but their thyrotropin-induced growth is insufficient, leading to congenital hypothyroidism in the vast majority of affected subjects. We hypothesized that arrested migration is due to premature differentiation [reflected by decreased telomere length (TL)], as observed in neural tube defects in mice. METHODS: Absolute TL and telomerase reverse transcriptase (hTERT) expression was measured in four ectopic and six orthotopic thyroids. TL was measured by quantitative polymerase chain reaction of genomic DNA, whereas hTERT expression was measured by quantitative polymerase chain reaction of total RNA. RESULTS: The mean±standard deviation TL (in kilobases per diploid genome) was 140.45±40.07 in ectopic and 97.50±30.48 in orthotopic thyroids (p=0.12). Expression of hTERT was quiescent in both ectopic and orthotopic thyroids. CONCLUSIONS: Compared with orthotopic thyroids, TL shortening is not observed in ectopic thyroid tissues and, consequently, no compensatory hTERT expression was measured. This makes premature differentiation an unlikely cause of arrested migration and it suggests, indirectly, that ectopic thyroids are not at higher risk of cancer than orthotopic thyroids.

Sublingual thyroid ectopy: similarities and differences with Kallmann syndrome.

Permanent primary congenital hypothyroidism (CH), the commonest cause of preventable intellectual disability, is due to defects in the embryonic development of the thyroid in the vast majority of cases. These defects are collectively called thyroid dysgenesis. The thyroid may be absent (athyreosis) but, more commonly, a sublingual thyroid ectopy without lateral lobes, is the only thyroid tissue present. Such an ectopy presumably results from an arrest in the downward migration of the median anlage. Thyroid ectopy almost always occurs in a sporadic fashion. However, first-degree relatives are affected more often than chance alone would predict. On the other hand, almost all reported monozygotic twin pairs are discordant for thyroid ectopy. Current research is aimed at reconciling these contradictory epidemiological data. We propose a two-hit mechanism associating a germline predisposing factor with another genetic or epigenetic alteration within the ectopic thyroid tissue itself or, as in some forms of Kallmann syndrome, in the structures surrounding the thyroid during embryogenesis. Thyroid ectopy, a model for sporadic congenital malformations in humans, is also associated with congenital heart disease, and molecular mechanisms common to thyroid and heart development are being unraveled.

Expression of CD133 in differentiated thyroid cancer of young patients.

AIMS: CD133 expression in cancer is frequently associated with poor outcome. Thyroid carcinomas are rare in childhood and adolescence and are associated with a higher risk of recurrence and more metastases than the adult tumours. The aim of the study was to assess whether the expression of CD133 in thyroid carcinomas of children, adolescents and young adults was correlated with clinical prognostic factors. METHODS: Tissue microarrays were constructed with 235 tumours coming from 208 young adults with a median age of 28 years and 27 children with a median age of 13 years. An immunohistochemical study was performed with anti-CD133 antibody. CD133 expression was evaluated, using a semiquantitative score based on the percentage of positive cells. The mutation status of tumours was evaluated by reverse transcriptase PCR. Three cell lines were used to confirm CD133 expression by western blot. RESULTS: CD133 expression was found in 43% of adult and 37% of child tumours and was confirmed by western blot in cell lines. In young adults, the expression of CD133 was significantly more frequent in patients with tumours >3 cm (p=0.04) and in patients with lymph node metastases (p=0.02). The expression of CD133 was more frequent in patients in whom the tumour presented a BRAF mutation (p=0.03). CONCLUSIONS: CD133 expression is correlated with tumour size, lymph nodes metastases and BRAF mutations in young adults. The presence of these cancer stem cells could offer new therapeutic alternatives for aggressive thyroid cancers.

Somatic mutations are not observed by exome sequencing of lymphocyte DNA from monozygotic twins discordant for congenital hypothyroidism due to thyroid dysgenesis.

BACKGROUND/AIMS: Congenital primary hypothyroidism (CH) is a rare pediatric disorder estimated to occur in about 1:2,500 live births. Approximately half of these cases entail ectopic thyroid tissue, which is believed to result from a migration defect during embryogenesis. Approximately 3% of CH cases are explained by mutation(s) in known genes, most of which are transcription factors implicated in the embryology of the thyroid gland. Surprisingly, monozygotic (MZ) twins are usually discordant for CH due to thyroid dysgenesis, suggesting that most cases are not caused by transmitted genetic variation. One possible explanation is somatic mutation in genes involved in thyroid migration occurring after zygotic twinning. Such mutations should be observed only in the affected twin. METHODS: To test the hypothesis of somatic mutation, we performed whole exome sequencing of DNA from three pairs of MZ twins discordant for CH with ectopic glands. RESULTS: We found no somatic mutations exclusive to any of the three affected twins or in any of the unaffected twins. CONCLUSION: Either somatic mutations are not significant for the etiology of CH or else such mutations lie outside regions of the genome accessible by exome sequencing technology.