Embryonic Thyroid Hormone Insufficiency Causes Structural Anomalies in the Embryo of Domestic Chick, Gallus domesticus.

Thyroid hormone (TH) is essential for growth and development, yet its specific role during embryogenesis remains incompletely understood. This study investigates the impact of TH deficiency, induced by thiourea, a known inhibitor of thyroid peroxidase (TPO), on the development of domestic chicks. Thiourea was administered before thyroid gland formation, and its presence in treated embryos was confirmed through liquid chromatography-mass spectrometry. In silico docking revealed a strong interaction between thiourea and the CCP-like domain of TPO, which was corroborated by TPO activity assays showing reduced enzyme function. This reduction in enzyme activity led to lower embryonic TH levels and increased thyroid-stimulating hormone (TSH) secretion. Morphological analysis of newly hatched chicks revealed significant structural anomalies, particularly in lateral plate mesoderm-derived structures, including omphalocele, limb deformities, anophthalmia and craniofacial defects. Alcian blue and Alizarin red staining demonstrated reduced ossification in ribs and forelimbs, while histological analysis showed incomplete abdominal wall closure and abnormal vertebral column development. Haematological profiling of TH-deficient newly hatched chicks revealed significantly lower blood cell counts, highlighting TH's critical role in haematopoiesis. These findings emphasise the multifaceted role of TH in embryonic development, with potential implications for understanding congenital hypothyroidism and its developmental impacts, especially in regions with limited healthcare access.

[Analysis of clinical characteristics and variant of NKX2-1 gene in a Chinese boy with Brain-Lung-Thyroid syndrome].

OBJECTIVE: To carry out clinical and genetic analysis for a child featuring Brain-Lung-Thyroid syndrome (BLTS). METHODS: A child who had presented at the Children's Hospital Affiliated to Shandong University on May 27, 2022 was selected as the study subject. Clinical data was collected. Trio-whole exome sequencing (Trio-WES) was carried out for the child and his parents, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. The child was given individualized treatment following the diagnosis. RESULTS: The child, a two-year-and-seven-month-old boy, had presented with global developmental delay, ataxia and hypothyroidism. WES revealed that he has harbored a heterozygous c.674C>T variant of the NKX2-1 gene, based on which he was diagnosed with BLTS. CT scan revealed interstitial and parenchymal inflammation in his lungs, which was reduced by budesonide aerosol inhalation. CONCLUSION: Discovery of the novel c.674C>T variant has enriched the mutational spectrum of the NKX2-1 gene. Budesonide aerosol may be used to treat lung inflammation associated with BLTS.

Patients with Thyroid Dyshormonogenesis and DUOX2 Variants: Molecular and Clinical Description and Genotype-Phenotype Correlation.

Thyroid dyshormonogenesis (THD) is a heterogeneous group of genetic diseases caused by the total or partial defect in the synthesis or secretion of thyroid hormones. Genetic variants in DUOX2 can cause partial to total iodination organification defects and clinical heterogeneity, from transient to permanent congenital hypothyroidism. The aim of this study was to undertake a molecular characterization and genotype-phenotype correlation in patients with THD and candidate variants in DUOX2. A total of 31 (19.38%) patients from the Catalan Neonatal Screening Program presented with variants in DUOX2 that could explain their phenotype. Fifteen (48.39%) patients were compound heterozygous, 10 (32.26%) heterozygous, and 4 (12.90%) homozygous. In addition, 8 (26.67%) of these patients presented variants in other genes. A total of 35 variants were described, 10 (28.57%) of these variants have not been previously reported in literature. The most frequent variant in our cohort was c.2895_2898del/p.(Phe966SerfsTer29), classified as pathogenic according to reported functional studies. The final diagnosis of this cohort was permanent THD in 21 patients and transient THD in 10, according to reevaluation and/or need for treatment with levothyroxine. A clear genotype-phenotype correlation could not be identified; therefore, functional studies are necessary to confirm the pathogenicity of the variants.

Time-resolved interactome profiling deconvolutes secretory protein quality control dynamics.

Many cellular processes are governed by protein-protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein-protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes.

Normal Values for the fT3/fT4 Ratio: Centile Charts (0-29 Years) and Their Application for the Differential Diagnosis of Children with Developmental Delay.

Primary congenital hypothyroidism is easily diagnosed on the basis of elevated plasma levels of thyroid-stimulating hormone (TSH). In contrast, in the rare disorders of thyroid hormone resistance, TSH and, in mild cases, also thyroid hormone levels are within the normal range. Thyroid hormone resistance is caused by defects in hormone metabolism, transport, or receptor activation and can have the same serious consequences for child development as congenital hypothyroidism. A total of n = 23,522 data points from a large cohort of children and young adults were used to generate normal values and sex-specific percentiles for the ratio of free triiodothyronine (T3) to free thyroxine (T4), the fT3/fT4 ratio. The aim was to determine whether individuals with developmental delay and genetically confirmed thyroid hormone resistance, carrying defects in Monocarboxylate Transporter 8 (MCT8), Thyroid Hormone Receptor alpha (THRα), and Selenocysteine Insertion Sequence-Binding Protein 2 (SECISBP2), had abnormal fT3/fT4 ratios. Indeed, we were able to demonstrate a clear separation of patient values for the fT3/fT4 ratio from normal and pathological controls (e.g., children with severe cerebral palsy). We therefore recommend using the fT3/fT4 ratio as a readily available screening parameter in children with developmental delay for the identification of thyroid hormone resistance syndromes. The fT3/fT4 ratio can be easily plotted on centile charts using our free online tool, which accepts various SI and non-SI units for fT3, fT4, and TSH.

A case report on congenital hypothyroidism and alpha thalassemia in children with anemia and muscle damage as the main manifestation.

RATIONALE: This study reports the first case of congenital hypothyroidism (CH) and alpha thalassemia in a child in China, with anemia and muscle damage as the main manifestations. Analyzing and studying this case is of great significance in reducing missed and misdiagnosed CH and will provide a clinical strategy for treating these patients. PATIENT CONCERNS: Child, female, 2 years and 7 months old, the child appeared dispirited, had poor appetite, shallow complexion, reduced activities with anemia, elevated muscle enzymes, height, and growth retardation. DIAGNOSES: The child was diagnosed with CH with alpha thalassemia. INTERVENTIONS: The patient was treated with levothyroxine sodium and anemia correction. OUTCOMES: The children's current spirit, appetite, red face, normal limb activity, physical development, and intelligence were significantly better than those of normal children of the same age. CONCLUSIONS: CH with alpha thalassemia, especially anemia and muscle damage as the main manifestations, has not been reported. Administration of levothyroxine sodium is effective in correcting anemia in patients with CH and alpha thalassemia. LESSON: Due to CH and alpha thalassemia, there are no specific symptoms and they are prone to missed diagnosis and misdiagnosis. Therefore, patients with anemia and elevated muscle enzyme levels should be routinely tested for thyroid function to diagnose them early and provide proper treatment to avoid negative consequences.

Congenital and acquired hypothyroidism: Temporal and spatial trends in France from 2014 to 2019.

PURPOSE: To assess the incidence of congenital hypothyroidism (CH) and acquired hypothyroidism (AH) between 2014 and 2019 in continental France. METHODS: New cases of CH and AH were identified using the French National Health Data System (Système Nationale des Données de Santé, SNDS). Temporal trends were studied using linear regression models. Spatial distributions were studied using Moran's global index (I) and the statistical method and local indicators of spatial association. RESULTS: The incidence of permanent CH in females increased by 8.9 % per year (2014: 36.9 [31.1-43.7] per 100,000 birth-years vs. 2019: 51 [43.9-59.3] per 100,000 birth-years, p < 0.01). The incidence of AH decreased between 2014 and 2019 for both females (2014: 535.7 [533.2-538.2] per 100,000 person-years vs 2019: 335.5 [333.6-337.4] per 100,000 person-years, p < 0.01) and males (2014: 197.5 [195.9-199] per 100,000 person-years vs 2019: 141.7 [140.4-142.9] per 100,000 person-years, p < 0.01). The incidence of hypothyroidism was high in the Nord-Pas-De-Calais and Lorraine regions (CH and AH). CONCLUSIONS: The incidence of permanent CH in females has increased over time. AH incidence decreased. It seems necessary to investigate environmental factors in the disparity of incidence distribution.

Genetic etiology in patients diagnosed with congenital hypothyroidism with new-generation sequencing: A single-center experience.

AIM: Congenital hypothyroidism (CH) is the most common endocrine disorder of the newborn; it is seen in every 3000-4000 births. Genetic features can guide treatment for patients with in situ glands. The present study aimed to contribute to the literature on CH variants and to show the benefit that genetic analysis can provide to patients in follow-up. METHOD: A total of 52 patients (47 families) diagnosed with CH were included in the study. Overall, 32 target genes involved in thyroid physiology were investigated by next-generation sequencing (NGS). RESULTS: In total, 29 (55 %) of the patients were male, and the rate of dysgenesis was 19.2 %. In this study, 29 of 52 patients had at least one variant in one gene involved in CH (n = 29, 33 different variants) (Including likely benign variants and variants of unknown significance). There were 21 patients (40.3 %) with gland in situ. The most common variant was DUOX2 (20 %). The second most common variants were those in the TPO and TG genes (15 % and 15 %, respectively); 41.1 % of these were variants of uncertain significance (VUS), 26.4 % were pathogenic, 23.5 % were likely benign, and 11.7 % were likely pathogenic. On the basis of their zygosity, we identified 73.5 % heterozygous, 17.6 % homozygous, and 8.9 % combined heterozygous variants. There were mutant variants in two genes in six patients and three in one patient. CONCLUSION: This study found a variant in 55 % of the patients and shed light on the etiology of some cases of CH. The frequency of VUS was high. Although variants were identified in this study, their implication in the etiology of CH is not certain and, for most of the patients, it is also not sufficient for explaining the pathology with the current state of knowledge.

Paediatric thyroid disease.

The spectrum of thyroid disorders presenting to paediatricians is different to that seen by adult physicians. Referrals reflect cases detected by the neonatal screening programme for congenital hypothyroidism and many of the inherited defects of thyroid hormone generation or action will be manifest in early life. Autoimmune thyroid disease can be particularly challenging to manage in the young and the potential impact of thyroid status on neurodevelopment and schooling are key considerations throughout childhood and adolescence.

Frequency of Mutations in the TPO Gene in Patients with Congenital Hypothyroidism Due to Dyshormonogenesis in Chile.

Background and Objectives: Congenital thyroid dyshormonogenesis is caused by alterations in the synthesis of thyroid hormones in a newborn. Additionally, 10 to 20% of these cases are hereditary, caused by defects in proteins involved in hormonal synthesis. One of the most common causes is mutations in the thyroid peroxidase (TPO) enzyme gene, an autosomal recessive disease. We aimed to detect mutations of the TPO gene in 12 Chilean patients with congenital hypothyroidism due to dyshormonogenesis (CHD) and to characterize these patients clinically and molecularly. Materials and Methods: Twelve patients under 20 years of age with CHD, controlled at San Juan de Dios Hospital in Santiago, Chile, were selected according to the inclusion criteria: elevated neonatal TSH, persistent hypothyroidism, and thyroid normotopic by imaging study. Those with deafness, Down syndrome, and central or transient congenital hypothyroidism were excluded. Blood samples were taken for DNA extraction, and the 17 exons and exon-intron junctions of the TPO gene were amplified by PCR. The PCR products were sequenced by Sanger. Results: Two possibly pathogenic mutations of the TPO gene were detected: c.2242G>A (p.Val748Met) and c.1103C>T (p.Pro368Leu). These mutations were detected in 2 of 12 patients (16.6%): 1 was compound heterozygous c.1103C>T/c.2242G>A, and the other was heterozygous for c.2242G>A. In the diagnostic confirmation test, both patients presented diffuse hyper-uptake goiter on thyroid scintigraphy and high TSH in venous blood (>190 uIU/mL). Conclusions: The frequency of patients with possibly pathogenic mutations in TPO with CHD was 16.6%. Its study would allow for genetic counseling to be offered to the families of affected patients.

Clinical efficacy of multigene panels in the management of congenital hypothyroidism with gland in situ.

Congenital hypothyroidism (CHT) is a diverse condition with various genetic etiologies. This study aimed to investigate the utility of next-generation sequencing (NGS) analysis in guiding treatment decisions and predicting prognosis for CHT patients with gland in situ (GIS). A retrospective analysis was conducted on 33 CHT patients with GIS who underwent NGS analysis at a single institution between 2018 and 2023. Patients were classified as having permanent (PCH), transient congenital hypothyroidism, or ambiguous congenital hypothyroidism (ACH) CHT based on their response to levothyroxine discontinuation at 3 years of age. Among the 33 patients, genetic variants were identified in 26, with the most prevalent variants found in DUOX2 (26.92%), TSHR (30.77%), TG (19.35%), and DUOXA2 (19.23%). Patients with high initial thyroid-stimulating hormone levels (>50 mIU/L) and low free thyroxine levels (<0.89 ng/dL) at diagnosis tended to have compound heterozygous or homozygous variants in DUOX2, DUOXA2, and TG, and were more likely to develop PCH. In contrast, patients with heterozygous variants in these genes often exhibited ACH. TSHR variants were associated with diverse clinical manifestations, ranging from PCH to ACH, and were more common in patients with initial thyroid-stimulating hormone levels <50 mIU/L. The study highlights the potential utility of NGS analysis in predicting the clinical course and guiding treatment decisions for CHT patients with GIS. Genetic analysis may aid in determining the appropriate duration of levothyroxine therapy and monitoring strategies, particularly in cases where traditional clinical indicators are inconclusive.

The longitudinal growth trajectory of children with congenital hypothyroidism during the first 3 years of life.

Congenital hypothyroidism (CH) is detected through a newborn screening program in Iran, enabling early detection and prompt treatment. This study addresses the longitudinal growth trajectory of Iranian children with CH and explores associated factors during the first 3 years of life. Data from 1474 children with CH in Isfahan, Iran (2002-2022), were analyzed. Weight, height, and head circumference were measured, and z-scores for age were calculated. Group-based trajectory modeling was applied to distinct growth trajectories. Factors influencing growth patterns, including gender, treatment initiation age, delivery method, parental consanguinity, history of familial hypothyroidism, and thyroid-stimulating hormone (TSH) levels at 3-7 days, were investigated. Thirty-seven percent of children diagnosed with CH faced a delay in weight, while 36.6% experienced stunted height, and 25.7% showed a retardation in head circumference growth. The initiation of treatment, parental consanguinity, and family history of hypothyroidism varied among these groups. Children exhibiting an optimal growth pattern in the initial 3 years of life demonstrated lower average TSH levels. CONCLUSION: This research emphasizes the complexity of managing CH and stresses the importance of tailoring interventions based on individualized characteristics and the ongoing growth patterns of the children. Future research is required to understand the intricate relationships between growth patterns and various determinants and optimize the growth and developmental outcomes of children with CH. WHAT IS KNOWN: • Iran has a higher prevalence of congenital hypothyroidism (CH) with a nationwide screening program. • There are concerns about delayed growth in CH children, but limited research on long-term patterns and contributing factors. WHAT IS NEW: • Distinct patterns in weight, height, and head circumference among children with CH were identified. • Factors such as consanguinity, parental hypothyroidism, and TSH levels impact growth outcomes. • CH management is complicated, and there is a need for individualized interventions.

Clinical and molecular study of patients with thyroid dyshormogenesis and variants in the thyroglobulin gene.

Introduction: Defects in any thyroid hormone synthesis steps cause thyroid dyshormonogenesis (THD). THD due to thyroglobulin (TG) gene variants is a cause of congenital hypothyroidism (CH) with a wide clinical spectrum, ranging from mild to severe permanent hypothyroidism. We present high-throughput sequencing results of patients with TG variants. Methods: A CH high-throughput sequencing-panel of the main genes involved in the regulation of thyroid hormonogenesis was performed to identify those TG variants that may be related to patient THD phenotype. Results: We identified 21 TG gene variants in 19 patients (11.8%) which could explain their phenotype. Ten of those (47.6%) were not previously described. CH was biochemically severe in these 19 patients. Eight of them were reevaluated after one month of discontinuing LT4 treatment and all had severe permanent hypothyroidism. We also identified another 16 patients who presented heterozygous TG variants, of whom, at reevaluation, five had mild permanent and only one had severe permanent hypothyroidisms. Discussions: In this study, 10 novel and 11 previously reported variants in the TG gene have been identified that could explain the phenotype of 19 patients from non-consanguineous families from a large THD cohort. Although not all these TG gene variants can explain all the patients' THD phenotypes, some of them had severe or mild permanent hypothyroidism at reevaluation.

Systematic review of thyroid function in NKX2-1-related disorders: Screening and diagnosis.

BACKGROUND: NKX2-1-related disorders (NKX2-1-RD) are rare conditions affecting lung, thyroid, and brain development, primarily caused by pathogenic variants or deletions in the NKX2-1 gene. Congenital hypothyroidism (CH) is a common endocrine manifestation, leading to irreversible intellectual disability if left untreated. OBJECTIVES: The aim was to evaluate the current evidence for the use of screening and diagnostic techniques for endocrine alterations in patients with NKX2-1-RD. METHODS: This systematic review was reported following the PRISMA guidelines. Two separate research questions in PICO format were addressed to cover initial screening and diagnosis procedures for endocrine diseases in patients with NKX2-1-RD. Eligibility criteria focused on patients with genetic confirmation of the disease and hypothyroidism. Various databases were searched, and data were extracted and assessed independently by two reviewers. RESULTS: Out of 1012 potentially relevant studies, 46 were included, for a total of 113 patients. CH was the most frequent endocrine alteration (45% of patients). Neonatal screening was reported in only 21% of patients based on blood TSH measurements. TSH thresholds varied widely across studies, making hypothyroidism detection ranges difficult to establish. Diagnostic tests using serum TSH were used to diagnose hypothyroidism or confirm its presence. 35% of patients were diagnosed at neonatal age, and 42% at adult age. Other hormonal dysfunctions identified due to clinical signs, such as anterior pituitary deficiencies, were detected later in life. Thyroid scintigraphy and ultrasonography allowed for the description of the thyroid gland in 30% of cases of hypothyroidism. Phenotypic variability was observed in individuals with the same variants, making genotype-phenotype correlations challenging. CONCLUSION: This review highlights the need for standardized protocols in endocrine screening for NKX2-1-RD, emphasizing the importance of consistent methodology and hormone threshold levels. Variability in NKX2-1 gene variants further complicates diagnostic efforts. Future research should concentrate on optimizing early screening protocols and diagnostic strategies.

Differential diagnosis of Huntington's disease- neurological aspects of NKX2-1-related disorders.

Benign hereditary chorea (BHC) is an inherited neurological disorder consisting of childhood-onset, nonprogressive chorea, generally without any other manifestations. In most reported cases, the inheritance of BHC is autosomal dominant but both incomplete penetrance and variable expressivity are observed and can be caused by NKX2-1 mutations. The spectrum contains choreoathetosis, congenital hypothyroidism, and neonatal respiratory distress syndrome. The neurological symptoms can be misdiagnosed as Huntington's disease (HD). The two Polish families were diagnosed with NKX2-1 gene mutations and a literature review concerning the NKX2-1-related disorders was conducted. All family members were examined by experienced movement disorders specialists. PubMed database was searched to obtain previously described NKX2-1 cases. Whole exome sequencing (WES) was performed in one proband (Family A) and direct NKX2-1 sequencing in the second (Family B). Two Polish families were diagnosed with NKX2-1 gene mutations (p.Trp208Leu and p.Cys117Alafs*8). In one family, the co-occurrence of HD was reported. Forty-nine publications were included in the literature review and symptoms of 195 patients with confirmed NKX2-1 mutation were analyzed. The most common symptoms were chorea and choreiform movements, and delayed motor milestones. The NKX2-1 mutation should always be considered as a potential diagnosis in families with chorea, even with a family history of HD. Lack of chorea does not exclude the NKX2-1-related disorders.

Identification of southern Taiwan genetic variants in thyroid dyshormonogenesis through whole-exome sequencing.

Thyroid dyshormonogenesis (TDH) is responsible for 15%-25% of congenital hypothyroidism (CH) cases. Pathogenetic variants of this common inherited endocrine disorders vary geographically. Unraveling the genetic underpinnings of TDH is essential for genetic counseling and precise therapeutic strategies. This study aims to identify genetic variants associated with TDH in Southern Taiwan using whole exome sequencing (WES). We included CH patients diagnosed through newborn screening at a tertiary medical center from 2011 to 2022. Permanent TDH was determined based on imaging evidence of bilateral thyroid structure and the requirement for continuous medication beyond 3 years of age. Genomic DNA extracted from blood was used for exome library construction, and pathogenic variants were detected using an in-house algorithm. Of the 876 CH patients reviewed, 121 were classified as permanent, with 47 (40%) confirmed as TDH. WES was conducted for 45 patients, and causative variants were identified in 32 patients (71.1%), including DUOX2 (15 cases), TG (8 cases), TSHR (7 cases), TPO (5 cases), and DUOXA2 (1 case). Recurrent variants included DUOX2 c.3329G>A, TSHR c.1349G>A, TG c.1348delT, and TPO c.2268dupT. We identified four novel variants based on genotype, including TSHR c.1135C>T, TSHR c.1349G>C, TG c.2461delA, and TG c.2459T>A. This study underscores the efficacy of WES in providing definitive molecular diagnoses for TDH. Molecular diagnoses are instrumental in genetic counseling, formulating treatment, and developing management strategies. Future research integrating larger population cohorts is vital to further elucidate the genetic landscape of TDH.

Incidence and associated risk factors of congenital hypothyroidism among newborns in Hainan, China: a retrospective study.

OBJECTIVES: This study aims to investigate the incidence and risk factors of congenital hypothyroidism (CH) in newborns in Hainan Province, China, to provide a reference for early and effective prevention strategies. METHODS: Newborns born in Hainan Province from 2017 to 2021 were the subjects of this study. Time-resolved immunofluorescence was used for initial screening and chemiluminescence for confirmatory diagnosis. Based on the diagnosis, newborns were classified into CH and non-CH groups. Statistical analysis was conducted on the initial screening and confirmed CH cases in newborns in Hainan Province, and potential risk factors for CH were explored. RESULTS: From 2017 to 2021, a total of 585,886 newborns were screened, revealing 6,856 initial positive results, 614 positive rescreens, and 420 confirmed CH cases, yielding an incidence rate of 1/1,395 (420/585,886). The annual initial positive screening rate of newborns in Hainan Province showed a rising trend from 2017 to 2021 (p=0.000). No significant differences were found regarding gender (p=0.400) and ethnicity (p=0.836). Multivariate logistic regression analysis indicated that residing in coastal areas, especially those with salt fields (OR=2.151, 95 % CI: 1.364-3.390), was risk factors for the development of CH in newborns. CONCLUSIONS: The incidence of CH in newborns showed a year-on-year increase in Hainan Province from 2017 to 2021. Residing in coastal areas, particularly those with salt fields, was identified as a risk factor for the development of CH.

Insights from European Reference Network for rare neurological disorders study surveys on diagnosis, treatment, and management of NKX2-1-related disorders.

BACKGROUND: NKX2-1-related disorder (NKX2-1-RD) is a rare disease characterized by a triad of primary hypothyroidism, neonatal respiratory distress, and neurological features, including chorea. OBJECTIVE: This study aimed to identify discrepancies in the management of NKX2-1-RD among European Union (EU) specialists. METHODS: The ERN-RND Chorea & Huntington disease group designed a survey to conduct a cross-sectional multicenter study on the management of NKX2-1-RD. Descriptive analysis was performed, and total responses are presented for each item. RESULTS: The study involved 23 experts from 13 EU countries with experience in evaluating hyperkinetic patients with NKX2-1-RD: 11 were adult specialists, and 12 were pediatric specialists. NKX2-1-RD diagnosis was made at different ages, with the most common initial symptoms being hypotonia and/or motor developmental delay (reported by 11 experts) and chorea (reported by 8 experts). Chorea involved various body parts and showed improvement as reported by 9 experts, stabilization by 12 experts, and worsening by 2 experts with age. The pharmacological treatment of chorea varied widely among the experts. Misdiagnosis was reported by 14 experts. NKX2-1 pathogenic variants or deletions were confirmed in >75 % of patients (reported by 12 experts). Pulmonary and endocrinology evaluations were requested by 7 and 12 experts, respectively. The management of psychiatric comorbidities also varied among the different experts. CONCLUSIONS: This study highlights the need for a clinical practice guideline for the management of NKX2-1-RD to ensure that patients across the EU receive consistent and appropriate care. Such a guideline would benefit both doctors and healthcare practitioners.

Molecular and Clinical Features of Congenital Hypothyroidism Due to Multiple DUOX2 Variants.

Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown functions. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not yet been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutational spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.