Identification of ZBTB26 as a Novel Risk Factor for Congenital Hypothyroidism

Congenital primary hypothyroidism (CH; OMIM 218700) is characterized by an impaired thyroid development, or dyshormonogenesis, and can lead to intellectual disability and growth retardation if untreated. Most of the children with congenital hypothyroidism present thyroid dysgenesis, a developmental anomaly of the thyroid. Various genes have been associated with thyroid dysgenesis, but all known genes together can only explain a small number of cases. To identify novel genetic causes for congenital hypothyroidism, we performed trio whole-exome sequencing in an affected newborn and his unaffected parents. A predicted damaging de novo missense mutation was identified in the ZBTB26 gene (Zinc Finger A and BTB Domain containing 26). An additional cohort screening of 156 individuals with congenital thyroid dysgenesis identified two additional ZBTB26 gene variants of unknown significance. To study the underlying disease mechanism, morpholino knock-down of zbtb26 in Xenopus laevis was carried out, which demonstrated significantly smaller thyroid anlagen in knock-down animals at tadpole stage. Marker genes expressed in thyroid tissue precursors also indicated a specific reduction in the Xenopus ortholog of human Paired-Box-Protein PAX8, a transcription factor required for thyroid development, which could be rescued by adding zbtb26. Pathway and network analysis indicated network links of ZBTB26 to PAX8 and other genes involved in thyroid genesis and function. GWAS associations of ZBTB26 were found with height. Together, our study added a novel genetic risk factor to the list of genes underlying congenital primary hypothyroidism and provides additional support that de novo mutations, together with inherited variants, might contribute to the genetic susceptibility to CH.

Further Evidence That Defects in Main Thyroid Dysgenesis-Related Genes Are an Uncommon Etiology for Primary Congenital Hypothyroidism in Mexican Patients: Report of Rare Variants in FOXE1, NKX2-5 and TSHR

Mexico shows a high birth prevalence of congenital hypothyroidism (CH) due to thyroid dysgenesis (TD). PAX8 defects underlie only 1% of these cases and NKX2-1 does not seem to be involved. Here, we analyzed other TD-related genes in 128 non-related Mexican patients (females 77.3%; 6 months to 16.6 years) with non-syndromic CH-TD diagnosis established by clinical evaluation, thyroid hormone serum profiling, and scintigraphy (74%) or ultrasonography (26%). We performed Sanger sequencing of FOXE1, NKX2-5, and TSHR and evaluated copy number variations (CNVs) in TSHR, FOXE1, PAX8, and NKX2-1 by multiplex ligation-dependent probe amplification. Odds ratios for TD risk were explored for FOXE1 polyalanine stretches [polyAla-rs71369530] in cases and controls (N = 116). Five rare missense changes cataloged as benign (NKX2-5:p.(Ala119Ser)-rs137852684), of unknown significance (FOXE1:p.(Ala335Gly)-rs543372757; TSHR:p.(Asp118Asn)-rs1414102266), and likely pathogenic (FOXE1:p.(Gly124Arg)-rs774035532; TSHR:p.(Trp422Arg)-rs746029360) accounted for 1.5% (N = 2/128) of clinically relevant genotypes (supported in part by protein modeling) in CH-TD. No CNVs were identified, nor did polyAla > 14 alanines in FOXE1 significantly protect against TD. The present and previously published data collectively show that small clinically relevant germline variants in PAX8, FOXE1, and TSHR are found in only a very small proportion (2.5%) of isolated CH-TD Mexican patients.

Further Evidence that Defects in mMain Thyroid Dysgenesis-Related Genes are an Uncommon Etiology for Primary Congenital Hypothyroidism in Mexican Patients: Report of Rare Variants in FOXE1, NKX2-5 and TSHR

Mexico shows a high birth prevalence of congenital hypothyroidism (CH) due to thyroid dysgenesis (TD). PAX8 defects underlie only 1% of these cases and NKX2-1 does not seem to be involved. Here, we analyzed other TD-related genes in 128 non-related Mexican patients (females 77.3%; 6 months to 16.6 years) with non-syndromic CH-TD diagnosis established by clinical evaluation, thyroid hormone serum profiling, and scintigraphy (74%) or ultrasonography (26%). We performed Sanger sequencing of FOXE1, NKX2-5, and TSHR and evaluated copy number variations (CNVs) in TSHR, FOXE1, PAX8, and NKX2-1 by multiplex ligation-dependent probe amplification. Odds ratios for TD risk were explored for FOXE1 polyalanine stretches [polyAla-rs71369530] in cases and controls (N=116). Five rare missense changes cataloged as benign (NKX2-5:p.(Ala119Ser)-rs137852684), of unknown significance (FOXE1:p.(Ala335Gly)-rs543372757; TSHR:p.(Asp118Asn)-rs1414102266), and likely pathogenic (FOXE1:p.(Gly124Arg)-rs774035532; TSHR:p.(Trp422Arg)-rs746029360) accounted for 1.5% (N=2/128) of clinically relevant genotypes (supported in part by protein modeling) in CH-TD. No CNVs were identified, nor did polyAla >14 alanines in FOXE1 significantly protect against TD. The present and previously published data collectively show that small clinically relevant germline variants in PAX8, FOXE1, and TSHR are found in only a very small proportion (2.5%) of isolated CH-TD Mexican patients.

Case Report: Primary Hypothyroidism Caused by Autoimmune Thyroiditis in Infancy Requires Early Intervention

Background: Primary hypothyroidism due to autoimmune thyroiditis is extremely rare in infants, especially under the age of 3 years. For infants, hypothyroidism is most commonly congenital, originating from thyroid dysgenesis with an absent, ectopic, or hypoplastic thyroid gland (1 in 4,000 live births). If left untreated, it can lead to permanent neurodevelopmental deficits. In this report, we describe a male infant who was diagnosed with Hashimoto thyroiditis at 18 months of life, providing a learning example to aid in recognition of this rare disease and enable timely intervention. Clinical Case:Patient was a 2,765 gram, appropriate for gestational age, male born at term with hypospadias of the penis (surgical correction at 11 months). Patient passed meconium in the first 24 hours of life. During the first few months of life, patient developed constipation. Patient had amblyopia necessitating eye patching and began to wear eye glasses at 18 months of life. Patient’s linear and weight growth were within normal limits. Patient had normal motor development, however had language development delay. No known family history of thyroid disease. Screening labs performed at 17-months of age showed abnormal thyroid function: elevated TSH at 14.86 µIU/mL (ref: 0.45 - 4.50 µIU/mL) and normal free T4 level at 1.24 ng/dL (ref: 0.85-1.75 ng/dL). Repeat testing at 18 months of age continued to show elevated TSH at 6.18 µIU/mL (ref: 0.64 - 4.00 µIU/mL), normal free T4 at 1.07 ng/dL (ref: 0.88 - 2.03 ng/dL), and elevated thyroid peroxidase (TPO) antibodies at 163 IU/ml (ref: <35 IU/ml). At 21 months of age, patient was initiated on L-thyroxine therapy for elevation of TSH (9.570 µIU/mL; ref: 0.64 - 4.00 µIU/mL) and free T4 was normal (1.03ng/dL; ref: 0.88 - 2.03 ng/dL). Notably, the newborn screen for hypothyroidism was within normal limits, suggesting chronic autoimmune thyroiditis instead of congenital thyroid dysgenesis. Conclusions: This case report provides insights into autoimmune thyroiditis in infancy, which, although especially rare under age 3 years, should be considered in infants who present with autoimmunity or abnormal thyroid testing. In the neonatal period, infants’ immune systems are learning to discriminate requirements for self-tolerance versus protection against pathogens and may be more prone to infections. Although autoimmunity in this stage of development is uncommon, there can be breakthroughs in tolerance, as seen in this case. In addition to this patient, two other infants were seen with elevated TPO antibodies, diagnosed at 17 and 31 months old, with similar clinical trends. There remains a need for additional studies providing further insights into autoimmunity in infancy. Importantly, this case illustrates that, when infants have abnormal thyroid levels (with or without other autoimmune conditions), consideration should be made for anti-thyroid antibody testing.

Two novel mutations in Gli-similar 3 in patients with congenital hypothyroidism and thyroid dysgenesis

Background: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH). As variants of the transcription factor Gli-similar 3 (GLIS3) have been associated with CH and GLIS3 is one of candidate genes of TD, we screened and characterized GLIS3 mutations in Chinese patients with CH and TD.Methods: To detect mutations, we sequenced all GLIS3 exons in the peripheral blood genomic DNA isolated from 50 patients with TD and 100 healthy individuals. Wild-type and mutant expression vectors of Glis3 were constructed. Quantitative real-time PCR, western blotting, and double luciferase assay were performed to investigation the effect of the mutations on GLIS3 protein function and transcriptional activation.Results: Two novel heterozygous missense mutations, c.2710G>A (p.G904R) and c.2507C>A (p.P836Q), were detected in two unrelated patients. Functional studies revealed that p.G904R expression was 59.95% lower and p.P836Q was 31.23% lower than wild-type GLIS3 mRNA expression. The p.G904R mutation also resulted in lower GLIS3 protein expression compared with that encoded by wild-type GLIS3. Additionally, the luciferase reporter assay revealed that p.G904R mediated impaired transcriptional activation compared with the wild-type protein (p < 0.05) but did not have a dominant-negative effect on the wild-type protein.Conclusions: We for the first time screened and characterized the function of GLIS3 mutations in Chinese individuals with CH and TD. Our study not only broadens the GLIS3 mutation spectrum, but also provides further evidence that GLIS3 defects cause TD.