Identification of a novel mutation in thyroxine-binding globulin (TBG) gene associated with TBG-deficiency and its effect on the thyroid function.

PURPOSE: This study presents a case of familial transmission of thyroxine-binding globulin (TBG) deficiency. The SERPINA7-gene which codes for TBG is located on the X-chromosome (Xq21-22). More than 45 mutations have been reported to cause TBG- deficiency from various countries, but none from India so far. Genetic analysis of SERPINA7 gene was carried out to determine the cause of low TBG levels in one family. METHODS: DNA samples of the propositus and the family members were subjected to Polymerase Chain Reaction (PCR) followed by direct sequencing. Allele-specific PCR and Next-gen sequencing (NGS) were employed to confirm the site of the mutation. Thyroid function tests were estimated by Radioimmunoassay (RIA) and Immunoradiometric assay (IRMA) kits. X-chromosomal inactivation status was analyzed in the female members harboring the mutation. RESULTS: A mutational screening in this family revealed a novel frame-shift mutation S353Q, 354fs3X in the exon 4 of the SERPINA7 gene which will be referred to as TBG-complete deficiency-India (TBG-CD-Ind). One out of four female family members harboring the mutation showed selective X-chromosomal inactivation. The affected family members were clinically euthyroid initially, showed changes in the thyroid function when tested after a long time span. However, the changes in the thyroid function in the affected family members had an autoimmune etiology. CONCLUSION: This study presents the first report of TBG-CD from India wherein a novel frameshift mutation referred to as TBG-CD-Ind (S353Q, 354fs3X) in the SERPINA7 gene was detected. No apparent association was identified between thyroid function and the TBG-mutation in the affected subjects. A detailed biochemical and genomic testing to determine the exact cause of discordant TFT in the patients would certainly aid in the unequivocal diagnosis of the thyroid function and for the precise individualized treatment.

A Newborn Falsely Suspected of Congenital Hypothyroidism due to Mutated Thyroxine-Binding Globulin with Low Binding Affinity.

INTRODUCTION: Neonatal screening programs for congenital hypothyroidism (CH) have been implemented worldwide to facilitate early diagnosis and treatment. The Dutch neonatal CH screening is primarily based on the measurement of thyroxine (T4). When T4 is low, an additional thyroxine-binding globulin (TBG) measurement is performed to reduce the number of false-positive screening results due to harmless TBG deficiency. Here, we present a case of a rare functional TBG deficiency leading to a false suspicion of CH. CASE PRESENTATION: Neonatal screening in this patient revealed a decreased T4, normal TSH, and normal TBG concentration, suggesting central CH. However, free T4 was normal. DNA sequencing analysis revealed a novel, hemizygous mutation (c.139G>A) in SERPINA7, the gene encoding TBG, resulting in the substitution of the conserved amino acid alanine to threonine at position 27. Crystal structure analyses showed that this substitution has a detrimental effect on binding of T4 to TBG. CONCLUSIONS: The novel SERPINA7 variant in this patient led to a false suspicion of central hypothyroidism in the Dutch T4-based neonatal screening program. It is important to recognize patients with such TBG defects to prevent unnecessary additional testing and treatment.

Compound hemizygous variants in SERPINA7 gene cause thyroxine-binding globulin deficiency.

SUB-HEADING: Compound hemizygous variants in SERPINA7 gene. BACKGROUND: Thyroxine-binding globulin (TBG) is encoded by SERPINA7 (OMIM. 314200) which is located on Xq22.3. SERPINA7 variants caused TBG deficiency which does not require treatment, but the decreased thyroxine may be misdiagnosed as hypothyroidism. We discovered some variants of TBG caused by alterations that differ from previously reported. MATERIALS AND METHODS: In this study, we enrolled 32 subjects from 10 families and sequenced the SERPINA7 genes of TBG-deficient subjects. Then, variants were analyzed to assess their effect on TBG expression and secretion. Bioinformatics database, protein structure, and dynamics simulation were used to evaluate the deleterious effects. Finally, we identified 2 novel and 4 known variants, and found 26 of 30 subjects carried the p.L303F. The DynaMut predictions indicated the variants (p.E91K, p.I92T, p.R294C, and p.L303F) exhibited decreased stability. CONCLUSION: Analyses revealed the p.L303F change the protein stability and flexibility, and it had an impact on the function of TBG, but when coexisted with other variants it might change the conformational structure of the protein and aggravate the damage to the protein. We speculated that the existence of a higher number of variants resulted in lower TBG secretion.

Partial thyroxine-binding globulin deficiency in a family with coding region mutations in the TBG gene.

PURPOSE: T4-binding globulin (TBG) is the main thyroid hormone (TH) transporter present in human serum. Inherited thyroxine-binding globulin (TBG) deficiency is caused by mutations in the TBG (SERPINA7) gene, which is located on the X chromosome. This study was performed to report and evaluate coding region mutations in TBG gene for partial thyroxine-binding globulin deficiency. METHODS: A pedigree spanning four generations is described in this study. The proband is a female with partial TBG deficiency. All members of this pedigree underwent thyroid function tests, while Sanger sequencing was used to identify the TBG gene mutations. Bioinformatics databases were used to evaluate the deleterious effects of the mutation(s). Two hundred and seven unrelated individuals were used to evaluate the thyroid function of individuals with different TBG mutations. A one-way ANOVA was used to analyze the impact of the TBG mutations on thyroid function. RESULTS: TBG gene sequencing results revealed that the proband had a novel mutation in codon 27 leading to alanine to valine substitution (p.A27V). This mutation was associated with lower serum T4 levels (p < 0.0001) when compared to the groups that did not carry the mutation. The previously reported p.L283F mutation was also found in the proband. The hemizygous p.L283F individuals presenting with lower T4 serum and TBG levels (p < 0.001) when compared to wildtype males and females. Both mutations were deleterious upon SIFT and PolyPhen-2 evaluation. CONCLUSION: Associated with partial thyroxine-binding globulin deficiency, this study reports a novel p.A27V mutation in the TBG gene.

Thyroxine-binding globulin deficiency due to a novel SERPINA7 mutation: Clinical characterization, analysis of X-chromosome inactivation pattern and protein structural modeling.

OBJECTIVE: Thyroxine-binding globulin (TBG) is the major human thyroid hormone transport protein, encoded by the SERPINA7 gene (Xq22.2). We aim to investigate the molecular basis of partial TBG deficiency (TBG-PD) in a female, by evaluating the X-chromosome inactivation pattern as well as the mutant protein structural modeling. DESIGN AND METHODS: Sequencing of the coding region of the SERPINA7 gene was performed in a female with a TBG-PD phenotype and her first-degree relatives. The proband presented with low serum levels of total T3 (TT3) and total T4 (TT4), serum TSH level of 5.4 µUI/mL (normal range, 0.35-5.5), and serum TBG level of 5.5 mg/L (normal range, 13.6-27.2). X-chromosome inactivation pattern was evaluated by methylation analysis of the androgen receptor gene (Xq11.2). Structural analysis of the SERPIN family was performed using Pymol and Areaimol, and PFSTATS for conservation analysis and family-wide investigation of equivalent positions in human homologs. Modeller was used for point mutation structural modeling. RESULTS: A novel missense SERPINA7 mutation (p.R35W; c.163C > T) was found in heterozygosity in the proband, and in hemizygosity in her affected siblings. The proband X-chromosome inactivation ratio was 20:80. The substitution of an arginine by a tryptophan is predicted to disrupt the protein surface and main electrostatic interactions. Tryptophans are extremely rare (0.1%) in this position. CONCLUSIONS: We report a new SERPINA7 variant associated with TBG-PD in three siblings. We named this variant TBG-Brasilia. The X-chromosome inactivation pattern may have accounted for the rare phenotypic expression in a female. The hydrophobic nature of the mutant is predicted to create an apolar patch at the surface, which results in protein aggregation and/or misfolding, potentially responsible for thyroid hormone transport defect.

TSH and FT4 Concentrations in Congenital Central Hypothyroidism and Mild Congenital Thyroidal Hypothyroidism.

Context: In central hypothyroidism (CeH), free thyroxine (FT4) concentrations are low, whereas thyrotropin (TSH) concentrations may be low, normal, or even slightly elevated due to reduced bioactivity. Congenital CeH (CCeH) may be isolated or part of multiple pituitary hormone deficiencies (MPHD). Objective: We tested our hypotheses that (1) TSH concentrations have a more U-shaped distribution in children with CCeH compared with children with a normally functioning hypothalamic-pituitary-thyroid axis and (2) TSH concentrations in children with CCeH with MPHD are higher compared with children with isolated CCeH. We also studied whether FT4 levels are helpful in distinguishing CCeH from mild congenital hypothyroidism of thyroidal origin (CH-T). Methods: Dutch neonatal screening TSH and first diagnostic TSH and FT4 were analyzed in all children diagnosed with permanent CCeH between 1995 and 2012. Controls were children with T4-binding globulin deficiency. FT4 concentrations in CCeH were compared with those in CH-T with TSH values in the same range as those of CCeH. Results: We studied 120 children with CCeH (isolated CCeH, n = 50; MPHD, n = 70) and 350 control subjects. Screening TSH concentrations were not significantly different (P = 0.055), but diagnostic TSH values were significantly different between the CCeH group and the control group (P = 0.037). TSH was significantly higher in MPHD compared with isolated CCeH (P = 0.004). FT4 concentrations were significantly lower in CCeH compared with mild CH-T (P < 0.0005). Conclusion: TSH values in CCeH have a more U-shaped distribution compared with controls with the highest TSH concentrations in MPHD. FT4 levels were significantly lower in CCeH compared with CH-T.

Detecting Congenital Central Hypothyroidism by Newborn Screening: Difficulty in Distinguishing from Congenital Thyroxine-Binding Globulin Deficiency.

BACKGROUND/AIMS: Congenital central hypothyroidism (CH-C) can be detected on newborn screening (NBS) by programs using thyroxine (T4)-reflex thyroid-stimulating hormone (TSH) test approach. CH-C must be distinguished from T4-binding globulin (TBG) deficiency. We sought to determine whether thyroid function tests reliably separate CH-C from TBG deficiency. METHODS: We analyzed NBS and serum free and total T4, T3 resin uptake (T3RU) or TBG, and TSH for infants in the Northwest Regional NBS Program (NWRSP) between the years 2008 and 2015 with either CH-C or TBG deficiency. RESULTS: We discovered a significant overlap in T3RU and TBG levels amongst 21 cases of CH-C and 250 cases of TBG deficiency. Mean serum TBG levels were lower in CH-C cases (20.3 µg/mL, range 14.2-33.3) than what is reported in healthy infants (28.6 µg/mL, range 19.1-44.6). Serum free T4 was lower in CH-C cases than TBG deficiency but did not always differentiate between the two conditions. CONCLUSION: CH-C benefits from detection on NBS but must be distinguished from TBG deficiency. The diagnosis of CH-C rests solely on subnormal serum free T4, but is supported by the demonstration of other pituitary hormone deficiencies. As an overlap exists, serum TBG (or T3RU) levels do not play a role in the diagnosis of CH-C.

Challenges in interpretation of thyroid hormone test results.

INTRODUCTION: In interpreting thyroid hormones results it is preferable to think of interference and changes in concentration of their carrier proteins. OUTLINE OF CASES: We present two patients with discrepancy between the results of thyroid function tests and clinical status. The first case presents a 62-year-old patient with a nodular goiter and Hashimoto thyroiditis. Thyroid function test showed low thyroid-stimulating hormone (TSH) and normal to low fT4. By determining thyroid status (TSH, T4, fT4,T3, fT3) in two laboratories, basal and after dilution, as well as thyroxine-binding globulin (TBG), it was concluded that the thyroid hormone levels were normal. The results were influenced by heterophile antibodies leading to a false lower TSH level and suspected secondary hypothyroidism.The second case, a 40-year-old patient, was examined and followed because of the variable size thyroid nodule and initially borderline elevated TSH, after which thyroid status showed low level of total thyroid hormones and normal TSH. Based on additional analysis it was concluded that low T4 and T3 were a result of low TBG. It is a hereditary genetic disorder with no clinical significance. CONCLUSION: Erroneous diagnosis of thyroid disorders and potentially harmful treatment could be avoided by proving the interference or TBG deficiency whenever there is a discrepancy between the thyroid function results and the clinical picture.

[Partial thyroxine binding globulin deficiency in test tube infants: report of cases and literature review].

OBJECTIVE: To investigate the clinical characteristics of twins with thyroxine binding globulin (TBG) deficiency and to find SERPINA7 gene mutations. METHOD: Data(2015) related to clinical characteristics, serum biochemistry, gene mutations and pedigree of two children with TBG deficiency were collected in the First Affiliated Hospital of College of Medicine, Zhejiang University. The related literature was searched form China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform, National Center for Biotechnology Information and PubMed (up to December 2015) by using search terms "Thyroxine binding globulin deficiency, gene, mutation" . RESULT: Both patients were diagnosed as central hypothyroidism at the beginning and treated with L-thyroxine. Both of the identical twins of the triplet were observed for mutation in exon3, c. 631Gï¹¥A(p.A211T), a new mutation had not been reported, but their parents and another non-identical triplet brother were normal. Literature review showed that 23 foreign cases with SERPINA7 gene mutation had been reported, however, no Chinese with SERPINA7 gene mutation had been reported. Among reported cases it was shown that SERPINA7 gene mutations located in exon, intron, promoter and enhancer. Up to now, 49 variants had been identified, 41 of them located in the mutated genes. Including these two cases, patients with thyroxine binding globulin deficiency were characterized by reduced serum TH levels, but normal free TH and TSH and absence of clinical manifestations. CONCLUSION: The new mutation of SERPINA7 gene c. 631Gï¹¥A(p.A211T)is not transmitted via the known X chromosome linked heredity, and as the cases were test tube triplet infants, it is a de novo mutation. The serum thyroid function tests of TBG deficiency showed decreased TT4, TT3 and normal TSH and TBG deficiency is often misdiagnosed as central hypothyroidism.

Novel mutation p.A64D in the Serpina7 gene as a cause of partial thyroxine-binding globulin deficiency associated with increases affinity in transthyretin by a known p.A109T mutation in the TTR gene.

Partial thyroxine-binding globulin deficiency (TBG-PD) is an endocrine defect with a prevalence of 1:4 000 in newborns. Due to the presence of a single TBG gene on the X chromosome, most familial TBG defects follow an X-linked inheritance pattern. Abnormal T4 binding to T4-binding prealbumin (TTR) is a rare cause of euthyroid hyperthyroxinemia, which is transmitted by autosomal dominant inheritance. The purpose of the present study was to identify and characterize new mutations in the Serpina7 and TTR genes in a complete family with typical TBG-PD. All patients underwent clinical and biochemical evaluation. Sequencing of DNA, population screening by (SSCP) analysis, and bioinformatics studies were performed. Molecular studies revealed a novel p.A64D mutation in the exon 1 of Serpina7 gene associated with the previously reported p.A109T mutation in the exon 4 of TTR gene. To our knowledge, this is the first report of a patient with a TBG-PD by a mutation in Serpina7 that was coincident with a mutation in TTR gene that increased affinity of TTR for T4. This work contributes to elucidate the molecular basis of the defects of thyroid hormone transport in serum and the improvement of the diagnosis avoiding unnecessary therapy.