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Detection of Novel Gene Variants Associated with Congenital Hypothyroidism in a Finnish Patient Cohort

View Article: PubMed Central - PubMed

ABSTRACT

Background:: Congenital hypothyroidism (CH) is defined as the lack of thyroid hormones at birth. Mutations in at least 15 different genes have been associated with this disease. While up to 20% of CH cases are hereditary, the majority of cases are sporadic with unknown etiology. Apart from a monogenic pattern of inheritance, multigenic mechanisms have been suggested to play a role in CH. The genetics of CH has not been studied in Finland so far. Therefore, multigenic sequencing of CH candidate genes was performed in a Finnish patient cohort with both familial and sporadic CH.

Methods:: A targeted next-generation sequencing (NGS) panel, covering all exons of the major CH genes, was applied for 15 patients with sporadic and 11 index cases with familial CH.

Results:: Among the familial cases, six pathogenic mutations were found in the TPO, PAX8, and TSHR genes. Furthermore, pathogenic NKX2.1 and TG mutations were identified from sporadic cases, together with likely pathogenic variants in the TG, NKX2.5, SLC26A4, and DUOX2 genes. All identified novel pathogenic mutations were confirmed by Sanger-sequencing and characterized in silico and/or in vitro.

Conclusion:: In summary, the CH panel provides an efficient, cost-effective, and multigenic screening tool for both known and novel CH gene mutations. Hence, it may be a useful method to identify accurately the genetic etiology for dyshormogenic, familial, or syndromic forms of CH.

No MeSH data available.


Characterization of the PAX8 R31C mutation in a familial CH case. (A) Sanger chromatogram visualizing the mutated allele in this family. (B) A loss of transactivation activity of the PAX8 R31C mutant compared with wild type using a TG promoter luciferase reporter assay. Bars represent means ± standard error of the mean from experiments performed on three separate days (n = 15; ****p ≤ 0.0001). (C) Three-dimensional PAX8 model with highlighted arginine at position 31. The crystallized PAX5 structure (backbone presentation green) together with a DNA response element (white backbone and translucent surface) was used to model PAX8 protein fragments. Examples of other known pathogenic PAX8 mutations are labeled with magenta sticks. Mutations at the hydrophobic inner core disturb the tight package between the helixes (such as Ile34Thr or Cys57Tyr). An intermolecular H-bond from Arg31 contacts the DNA and mediates the proper justification of the protein and the DNA toward each other.
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f3: Characterization of the PAX8 R31C mutation in a familial CH case. (A) Sanger chromatogram visualizing the mutated allele in this family. (B) A loss of transactivation activity of the PAX8 R31C mutant compared with wild type using a TG promoter luciferase reporter assay. Bars represent means ± standard error of the mean from experiments performed on three separate days (n = 15; ****p ≤ 0.0001). (C) Three-dimensional PAX8 model with highlighted arginine at position 31. The crystallized PAX5 structure (backbone presentation green) together with a DNA response element (white backbone and translucent surface) was used to model PAX8 protein fragments. Examples of other known pathogenic PAX8 mutations are labeled with magenta sticks. Mutations at the hydrophobic inner core disturb the tight package between the helixes (such as Ile34Thr or Cys57Tyr). An intermolecular H-bond from Arg31 contacts the DNA and mediates the proper justification of the protein and the DNA toward each other.

Mentions: Family F11 (Fig. 1) included two children with CH. Both affected individuals were found to carry a heterozygous PAX8 c.91C>T, p.R31C mutation causing an amino acid change in the PAX8 protein (Table 2). The detected arginine to cysteine mutation at position 31 leads to a loss of structural–functional constraints and intermolecular contacts to the DNA response element and thus a loss of functionality (Fig. 3). Both cases showed serum TSH levels >200 mIU/L, fT4 was in the low range of the reference values, and they had a small or hypoplastic thyroid gland evaluated by ultrasound. In addition, one of the affected patients had a right kidney agenesis. The father was diagnosed with nonautoimmune hypothyroidism at 30 years of age, and sequencing of PAX8 revealed that he was also a carrier of the PAX8 c.91C>T mutation. The mother and other siblings had normal TFTs and no alteration in the PAX8 sequence. In line with the modeling, the in vitro tests demonstrated that the transcriptional activity of the PAX8 mutant was almost completely lost compared with the WT protein (Fig. 3).


Detection of Novel Gene Variants Associated with Congenital Hypothyroidism in a Finnish Patient Cohort
Characterization of the PAX8 R31C mutation in a familial CH case. (A) Sanger chromatogram visualizing the mutated allele in this family. (B) A loss of transactivation activity of the PAX8 R31C mutant compared with wild type using a TG promoter luciferase reporter assay. Bars represent means ± standard error of the mean from experiments performed on three separate days (n = 15; ****p ≤ 0.0001). (C) Three-dimensional PAX8 model with highlighted arginine at position 31. The crystallized PAX5 structure (backbone presentation green) together with a DNA response element (white backbone and translucent surface) was used to model PAX8 protein fragments. Examples of other known pathogenic PAX8 mutations are labeled with magenta sticks. Mutations at the hydrophobic inner core disturb the tight package between the helixes (such as Ile34Thr or Cys57Tyr). An intermolecular H-bond from Arg31 contacts the DNA and mediates the proper justification of the protein and the DNA toward each other.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036323&req=5

f3: Characterization of the PAX8 R31C mutation in a familial CH case. (A) Sanger chromatogram visualizing the mutated allele in this family. (B) A loss of transactivation activity of the PAX8 R31C mutant compared with wild type using a TG promoter luciferase reporter assay. Bars represent means ± standard error of the mean from experiments performed on three separate days (n = 15; ****p ≤ 0.0001). (C) Three-dimensional PAX8 model with highlighted arginine at position 31. The crystallized PAX5 structure (backbone presentation green) together with a DNA response element (white backbone and translucent surface) was used to model PAX8 protein fragments. Examples of other known pathogenic PAX8 mutations are labeled with magenta sticks. Mutations at the hydrophobic inner core disturb the tight package between the helixes (such as Ile34Thr or Cys57Tyr). An intermolecular H-bond from Arg31 contacts the DNA and mediates the proper justification of the protein and the DNA toward each other.
Mentions: Family F11 (Fig. 1) included two children with CH. Both affected individuals were found to carry a heterozygous PAX8 c.91C>T, p.R31C mutation causing an amino acid change in the PAX8 protein (Table 2). The detected arginine to cysteine mutation at position 31 leads to a loss of structural–functional constraints and intermolecular contacts to the DNA response element and thus a loss of functionality (Fig. 3). Both cases showed serum TSH levels >200 mIU/L, fT4 was in the low range of the reference values, and they had a small or hypoplastic thyroid gland evaluated by ultrasound. In addition, one of the affected patients had a right kidney agenesis. The father was diagnosed with nonautoimmune hypothyroidism at 30 years of age, and sequencing of PAX8 revealed that he was also a carrier of the PAX8 c.91C>T mutation. The mother and other siblings had normal TFTs and no alteration in the PAX8 sequence. In line with the modeling, the in vitro tests demonstrated that the transcriptional activity of the PAX8 mutant was almost completely lost compared with the WT protein (Fig. 3).

View Article: PubMed Central - PubMed

ABSTRACT

Background:: Congenital hypothyroidism (CH) is defined as the lack of thyroid hormones at birth. Mutations in at least 15 different genes have been associated with this disease. While up to 20% of CH cases are hereditary, the majority of cases are sporadic with unknown etiology. Apart from a monogenic pattern of inheritance, multigenic mechanisms have been suggested to play a role in CH. The genetics of CH has not been studied in Finland so far. Therefore, multigenic sequencing of CH candidate genes was performed in a Finnish patient cohort with both familial and sporadic CH.

Methods:: A targeted next-generation sequencing (NGS) panel, covering all exons of the major CH genes, was applied for 15 patients with sporadic and 11 index cases with familial CH.

Results:: Among the familial cases, six pathogenic mutations were found in the TPO, PAX8, and TSHR genes. Furthermore, pathogenic NKX2.1 and TG mutations were identified from sporadic cases, together with likely pathogenic variants in the TG, NKX2.5, SLC26A4, and DUOX2 genes. All identified novel pathogenic mutations were confirmed by Sanger-sequencing and characterized in silico and/or in vitro.

Conclusion:: In summary, the CH panel provides an efficient, cost-effective, and multigenic screening tool for both known and novel CH gene mutations. Hence, it may be a useful method to identify accurately the genetic etiology for dyshormogenic, familial, or syndromic forms of CH.

No MeSH data available.