Characterization of a Novel Loss of Function Mutation of Pax8 Associated with Congenital Hypothyroidism

IEOS, G. Salvatore National Research Council, Naples, Italy.
Clinical Endocrinology (Impact Factor: 3.46). 12/2010; 73(6):808-14. DOI: 10.1111/j.1365-2265.2010.03851.x
Source: PubMed


Congenital hypothyroidism (CH) is a common endocrine disease that occurs in about 1:3000 newborns. In 80-85% of the cases, CH is presumably secondary to thyroid dysgenesis (TD), a defect in the organogenesis of the gland leading to an ectopic (30-45%), absent (agenesis, 35-40%) or hypoplastic (5%) thyroid gland. The pathogenesis of TD is still largely unknown. Most cases of TD are sporadic, although familial occurrences have occasionally been described. Recently, mutations in the PAX8 transcription factor have been identified in patients with TD.
Our aim was to identify and functionally characterize novel PAX8 mutations with autosomal dominant transmission responsible for TD.
The PAX8 gene was sequenced in a mother and child both suffering from congenital hypothyroidism (CH) because of thyroid hypoplasia. Subsequently, expression vectors encoding the mutated PAX8 were generated, and the effects of the mutation on both the DNA-binding capability and the transcriptional activity were evaluated.
PAX8 gene sequencing revealed a heterozygous mutation that consists of the substitution of a histidine residue with a glutamine at position 55 of the PAX8 protein (H55Q). When tested in cotransfection experiments with a thyroglobulin promoter reporter construct, the mutant protein turned out to be still able to bind DNA in Electrophoretic Mobility Shift Assay assays but transcriptionally inactive.
Our findings confirm the important role of PAX8 in normal thyroid development and support the evidence that in humans haploinsufficiency of PAX8 is associated with TD.

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    • "In the left side: DHPLC profiles of wild type (green) and mutated (brown) PAX8 exon 5. The partial denaturing temperature was set at 61.3°C and the chromatographic parameters were obtained by means of the WAVEMaker software (Transgenomic, Omaha, NE), based on the amplicon sequence [23]. In the right side: Sequencing electropherogram of exon 5. "
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    ABSTRACT: Background Congenital hypothyroidism is often secondary to thyroid dysgenesis, including thyroid agenesis, hypoplasia, ectopic thyroid tissue or cysts. Loss of function mutations in TSHR, PAX8, NKX2.1, NKX2.5 and FOXE1 genes are responsible for some forms of inherited congenital hypothyroidism, with or without hypoplastic thyroid. The aim of this study was to analyse the PAX8 gene sequence in several members of the same family in order to understand whether the variable phenotypic expression, ranging from congenital hypothyroidism with thyroid hypoplasia to mild subclinical hypothyroidism, could be associated to the genetic variant in the PAX8 gene, detected in the proband. Methods We screened a hypothyroid child with thyroid hypoplasia for mutations in PAX8, TSHR, NKX2.1, NKX2.5 and FOXE1 genes. We studied the inheritance of the new variant R133W detected in the PAX8 gene in the proband’s family, and we looked for the same substitution in 115 Caucasian European subjects and in 26 hypothyroid children. Functional studies were performed to assess the in vitro effect of the newly identified PAX8 gene variant. Results A new heterozygous nucleotide substitution was detected in the PAX8 DNA-binding motif (c.397C/T, R133W) in the proband, affected by congenital hypothyroidism with thyroid hypoplasia, in his older sister, displaying a subclinical hypothyroidism associated with thyroid hypoplasia and thyroid nodules, in his father, affected by hypothyroidism with thyroid hypoplasia and thyroid nodules, and his first cousin as well, who revealed only a subclinical hypothyroidism. Functional studies of R133W-PAX8 in the HEK293 cells showed activation of the TG promoter comparable to the wild-type PAX8. Conclusions In vitro data do not prove that R133W-PAX8 is directly involved in the development of the thyroid phenotypes reported for family members carrying the substitution. However, it is reasonable to conceive that, in the cases of transcriptions factors, such as Pax8, which establish several interactions in different protein complexes, genetic variants could have an impact in vivo.
    Full-text · Article · Aug 2014 · BMC Endocrine Disorders
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    • "PAX8 mutations and inactivation are implicated in various thyroid conditions. Congenital hypothyroidism is caused by several genetic defects and among these there are mutations in the PAX8 gene [1], [11], [12]. In addition to hypothyroidism, PAX8 plays a role also in the progression of follicular thyroid carcinomas and adenomas [13], [14]. "
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    ABSTRACT: The differentiation program of thyroid follicular cells (TFCs), by far the most abundant cell population of the thyroid gland, relies on the interplay between sequence-specific transcription factors and transcriptional coregulators with the basal transcriptional machinery of the cell. However, the molecular mechanisms leading to the fully differentiated thyrocyte are still the object of intense study. The transcription factor Pax8, a member of the Paired-box gene family, has been demonstrated to be a critical regulator required for proper development and differentiation of thyroid follicular cells. Despite being Pax8 well-characterized with respect to its role in regulating genes involved in thyroid differentiation, genomics approaches aiming at the identification of additional Pax8 targets are lacking and the biological pathways controlled by this transcription factor are largely unknown. To identify unique downstream targets of Pax8, we investigated the genome-wide effect of Pax8 silencing comparing the transcriptome of silenced versus normal differentiated FRTL-5 thyroid cells. In total, 2815 genes were found modulated 72 h after Pax8 RNAi, induced or repressed. Genes previously reported to be regulated by Pax8 in FRTL-5 cells were confirmed. In addition, novel targets genes involved in functional processes such as DNA replication, anion transport, kinase activity, apoptosis and cellular processes were newly identified. Transcriptome analysis highlighted that Pax8 is a key molecule for thyroid morphogenesis and differentiation. This is the first large-scale study aimed at the identification of new genes regulated by Pax8, a master regulator of thyroid development and differentiation. The biological pathways and target genes controlled by Pax8 will have considerable importance to understand thyroid disease progression as well as to set up novel therapeutic strategies.
    Full-text · Article · Sep 2011 · PLoS ONE
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    ABSTRACT: Screening of the known candidate genes involved in thyroid organogenesis has revealed mutations in a small subset of patients with congenital hypothyroidism due to thyroid dysgenesis (TD). We studied a girl with TD who had mutations in two transcription factors involved in thyroid development. Sequencing analysis of candidate genes involved in thyroid gland development revealed a new paternally inherited heterozygous mutation in the NKX2.5 gene (S265R) and a new maternally inherited heterozygous mutation in the PAX8 promoter region (-456C>T). Both parents and a brother, who was also heterozygous for both mutations, were phenotypically normal. Immunofluorescence microscopy showed a correct nuclear localization of both wild-type (WT) and mutant NKX2.5 proteins. EMSA demonstrated that the mutant NKX2.5 binds to the NKE_2, DIO2, TG, and TPO promoter elements equally well as the WT protein. However, the mutant NKX2.5 protein showed a 30-40% reduced transactivation of the thyroglobulin and the thyroid peroxidase promoters and a dominant-negative effect of the mutant NKX2.5. EMSA studies of the WT and mutant PAX8 promoter sequences incubated with nuclear extracts from PCCL3 cells exhibited a loss of protein binding capacity of the mutant promoter. In addition, the mutant PAX8 promoter showed a significantly reduced transcriptional activation of a luciferase reporter gene in vitro. Thus, this promoter mutation is expected to lead to reduced PAX8 expression. We identified new heterozygous mutations in both NKX2.5 and PAX8 genes of a girl with TD. Both defects might contribute to the phenotype.
    Preview · Article · Mar 2011 · The Journal of Clinical Endocrinology and Metabolism
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