The Severity of Congenital Hypothyroidism With Gland-In-Situ Predicts Molecular Yield by Targeted Next-Generation Sequencing.

INTRODUCTION: Congenital hypothyroidism with gland-in-situ (CH-GIS) is usually attributed to mutations in the genes involved in thyroid hormone production. The diagnostic yield of targeted next-generation sequencing (NGS) varied widely between studies. We hypothesized that the molecular yield of targeted NGS would depend on the severity of CH. METHODS: Targeted NGS was performed in 103 CH-GIS patients from the French national screening program referred to the Reference Center for Rare Thyroid Diseases of Angers University Hospital. The custom targeted NGS panel contained 48 genes. Cases were classified as solved or probably solved depending on the known inheritance of the gene, the classification of the variants according to the American College of Medical Genetics and Genomics, the familial segregation, and published functional studies. Thyroid-stimulating hormone at CH screening and at diagnosis (TSHsc and TSHdg) and free T4 at diagnosis (FT4dg) were recorded. RESULTS: NGS identified 95 variants in 10 genes in 73 of the 103 patients, resulting in 25 solved cases and 18 probably solved cases. They were mainly due to mutations in the TG (n = 20) and TPO (n = 15) genes. The molecular yield was, respectively, 73% and 25% if TSHsc was ≥ and < 80 mUI/L, 60% and 30% if TSHdg was ≥ and < 100 mUI/L, and 69% and 29% if FT4dg was ≤ and > 5 pmol/L. CONCLUSION: NGS in patients with CH-GIS in France found a molecular explanation in 42% of the cases, increasing to 70% when TSHsc was ≥ 80 mUI/L or FT4dg was ≤ 5 pmol/L.

Mutation screening of eight genes and comparison of the clinical data in a Chinese cohort with congenital hypothyroidism.

BACKGROUND: Congenital hypothyroidism (CH) is a common neonatal endocrine disorder, characterized by irreversible intellectual disability and short stature if left untreated. It can be divided into thyroid dysgenesis (TD), including athyreosis, ectopy and hypoplasia, and dyshormonogenesis (DH), also referring to gland in situ (GIS), in which patients have eutopic thyroids with normal size or goiter. This study aims to analyze the clinical and genetic data of 375 Chinese CH patients without DUOX2 and thyroid transcription factor (TTF) variants, and to explore the mutation frequencies of the eight genes and the inheritance pattern of CH. METHODS: Targeted next generation sequencing (NGS) and statistical analysis were performed for mutation screening on eight CH-related genes and the comparison of clinical data in a cohort of 606 Chinese CH patients from Henan Province. RESULTS: A total of 104 variants were detected in genes required for thyroid formation (TSHR, GLIS3, BOREALIN, NTN1, JAG1 and TUBB1) and thyroid hormone synthesis (TG and TPO) in 83 subjects. Monogenic variants were the most prevalent with a percentage of 75.00% (78/104) followed by oligogenic variants (25.00%, 26/104). No differences were found in various clinical data between patients with and without variants. However, it should be noted that only initial L-T4 dose was statistically different between patients with monogenic variants and oligogenic variants. CONCLUSIONS: Our results suggested that apart from Mendelian monogenic inheritance, oligogenic inheritance of CH could not be excluded and also involves other factors, such as penetrance, epigenetic mechanisms and environmental factors.

Functional analysis of PAX8 variants identified in patients with congenital hypothyroidism in situ.

Paired box transcription factor 8 (PAX8) is essential for thyroid organogenesis and development. Heterozygous pathogenic variants of PAX8 typically cause congenital hypothyroidism (CH) due to thyroid hypoplasia. Additionally, pathogenic PAX8 variants have been identified in patients with gland in situ (GIS). This study was conducted to analyze the in vitro functional consequences of four PAX8 variants (p.D94N, p.E90del, p.V58I, and p.L186Hfs*22) previously identified in patients with CH and GIS. The transcriptional activity of PAX8 variants on the thyroglobulin (TG) promoter was assessed in a luciferase reporter assay. The levels of transcriptional activity on the TG promoter of p.E90del and p.L186Hfs*22 were significantly reduced, whereas p.D94N and p.V58I showed residual activation. In addition, a dominant negative effect on the wild-type (WT) was not detected in any PAX8 variant using a luciferase reporter assay. Two PAX8 variants (p.E90del and p.L186Hfs*22) may be pathogenic causes of CH with GIS.

Molecular and clinical characteristics of congenital hypothyroidism in a large cohort study based on comprehensive thyroid transcription factor mutation screening in Henan.

BACKGROUND: Congenital hypothyroidism (CH), the most common neonatal endocrine disorder worldwide, can be caused by variants in thyroid transcription factor (TTF) genes including NKX2-1, FOXE1, PAX8, NKX2-5 and HHEX. This study aims to perform targeted next-generation sequencing (NGS) panel for comprehensive mutation screening on these genes in a cohort of 606 CH patients with various types from Henan Province, China, to investigate the mutation rate of TTF genes, and to analyze the clinical, biochemical and molecular characteristics of our CH cohort. METHODS: High-throughput sequencing combined with statistical calculation were applied for mutation screening and analyses of the clinical data. RESULTS: Twenty-two likely disease-causing monoallelic mutations in the TTF genes were identified in our cohort (3.63%, 22/606). Mutated PAX8 was the most predominant genetic alteration among these TTF mutations. Interestingly, PAX8 defects were only found in TD cases and variants in the five TTF genes were detected in gland in situ (GIS) patients. CH patients with the same genotype may have significant phenotypic variability and permanent CH (PCH) patients in the GIS group were significantly fewer than those in the TD group. CONCLUSIONS: Our study showed the estimated TTF mutation rate among CH cases was 3.63% in Henan Province and genetic alternations in TTF genes played a role not only in TD but also in GIS, especially in goiter. Although we speculated that the five TTF genes may be involved in certain steps of thyroid hormone biosynthesis, more researches are needed to verify the conclusions of the present study.

DUOX2 and DUOXA2 Variants Confer Susceptibility to Thyroid Dysgenesis and Gland-in-situ With Congenital Hypothyroidism.

Background: Thyroid dysgenesis (TD), which is caused by gland developmental abnormalities, is the most common cause of congenital hypothyroidism (CH). In addition, advances in diagnostic techniques have facilitated the identification of mild CH patients with a gland-in-situ (GIS) with normal thyroid morphology. Therefore, TD and GIS account for the vast majority of CH cases. Methods: Sixteen known genes to be related to CH were sequenced and screened for variations by next-generation sequencing (NGS) in a cohort of 377 CH cases, including 288 TD cases and 89 GIS cases. Results: In our CH cohort, we found that DUOX2 (21.22%) was the most commonly variant pathogenic gene, while DUOXA2 was prominent in TD (18.75%) and DUOX2 was prominent in GIS (34.83%). Both biallelic and triple variants of DUOX2 were found to be most common in children with TD and children with GIS. The most frequent combination was DUOX2 with DUOXA1 among the 61 patients who carried digenic variants. We also found for the first time that biallelic TG, DUOXA2, and DUOXA1 variants participate in the pathogenesis of TD. In addition, the variant p.Y246X in DUOXA2 was the most common variant hotspot, with 58 novel variants identified in our study. Conclusion: We meticulously described the types and characteristics of variants from sixteen known gene in children with TD and GIS in the Chinese population, suggesting that DUOXA2 and DUOX2 variants may confer susceptibility to TD and GIS via polygenic inheritance and multiple factors, which further expands the genotype-phenotype spectrum of CH in China.

High Diagnostic Yield of Targeted Next-Generation Sequencing in a Cohort of Patients With Congenital Hypothyroidism Due to Dyshormonogenesis.

Objective: To elucidate the molecular cause in a well-characterized cohort of patients with Congenital Hypothyroidism (CH) and Dyshormonogenesis (DH) by using targeted next-generation sequencing (TNGS). Study design: We studied 19 well-characterized patients diagnosed with CH and DH by targeted NGS including genes involved in thyroid hormone production. The pathogenicity of novel mutations was assessed based on in silico prediction tool results, functional studies when possible, variant location in important protein domains, and a review of the recent literature. Results: TNGS with variant prioritization and detailed assessment identified likely disease-causing mutations in 10 patients (53%). Monogenic defects most often involved TG, followed by DUOXA2, DUOX2, and NIS and were usually homozygous or compound heterozygous. Our review shows the importance of the detailed phenotypic description of patients and accurate analysis of variants to provide a molecular diagnosis. Conclusions: In a clinically well-characterized cohort, TNGS had a diagnostic yield of 53%, in accordance with previous studies using a similar strategy. TG mutations were the most common genetic defect. TNGS identified gene mutations causing DH, thereby providing a rapid and cost-effective genetic diagnosis in patients with CH due to DH.

Screening for congenital hypothyroidism: a worldwide view of strategies.

Detection by newborn screening (NBS) and treatment of babies with congenital hypothyroidism (CH) has largely eliminated the intellectual disability caused by this disorder. Lowering of the screening TSH cutoff and changes in birth demographics have been associated with an approximate doubling of the incidence of CH, from 1:3500 to 1:1714. The additional cases detected by lowering of the TSH cutoff tend to have milder hypothyroidism, with imaging often demonstrating a eutopic, "gland in-situ", and some cases turn out to have transient CH. Based on our search for current screening programs, approximately 71 percent of babies worldwide are not born in an area with an established NBS program, despite the existence of screening for over five decades in developed countries. Thus, the majority of babies with CH worldwide are not detected and treated early, such that the economic burden of retardation owing to CH remains a significant public health challenge.