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

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.

FIG. 1.

Modified pedigrees and identified mutations of the familial congenital hypothyroidism (CH) cases with genetic and clinical data. The results of the thyroid function tests and thyroid size are aligned below. The reference values of umbilical blood thyrotropin (uTSH), and thyrotropin (TSH) and free thyroxine (fT4) serum hormone levels at three days of age or the time of diagnosis are included. Black, affected patients with CH; symbols with midline, asymptomatic heterozygous carriers; gray, adult onset hypothyroidism; white, no thyroid disease. Lined box (case 5) indicates the TPO R438H mutation. Serum thyroglobulin (TG) levels were measured at the time of enrollment into the study. The age (years) at time of diagnosis (0 = at birth) is shown. Thyroid size evaluated by thyroid ultrasound if data available (n, normal thyroid size and location; +, goiter; a, athyreosis/no thyroid gland not detected; h, hypoplastic or small thyroid; n*, no clinical signs of goiter during follow-up; #, no DNA available). The detailed clinical information of the families is described in the Supplementary Materials and Methods. NA, data not available. ^aPatient had mild hypotonia, delay of development (speech and motor development) and abnormal hearing response, ^babnormal hearing, ^cborn in week 29 + 3, or ^drenal agenesis.

FIG. 2.

Confirmation of the TPO mutations identified from familial cases by Sanger sequencing and protein modeling. Sanger chromatograms of (A) the heterozygous (HET) and homozygous (HOM) TPO c.1182_1183insCGGC mutant and (B) TPO c.1313G>A point mutation compared with WT sequence. (C) A schematic picture of TPO with the localization of the mutations. (D) Dimeric model of the myeloperoxidase-like domain of TPO based on a homologous dimeric structure of myeloperoxidase. The homology model contains residues from Cys146-Thr735, whereby only the backbone is visualized (ribbon-tube cartoon). The TPO protomers are colored differently (the heme group, ions, and glycosylations are not represented). Cysteine disulfide-bridges (yellow sticks) are involved in maintaining the quaternary structure. TM, transmembrane domain. The side-chain of arginine at position 438 is connected via an H-bond to the loop-backbone at proline 153 and functions in consequence as a structural constraint between both domain fragments. The histidine mutant at position 438 likely fails establishing this important intramolecular interaction and leads to modification of the structural adjustment inside the domain.

FIG. 3.

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.