Comprehensive Screening of Eight Known Causative Genes in Congenital Hypothyroidism With Gland-in-Situ

Abstract

Context: Lower TSH screening cutoffs have doubled the ascertainment of congenital hypothyroidism (CH), particularly cases with a eutopically located gland-in-situ (GIS). Although mutations in known dyshormonogenesis genes or TSHR underlie some cases of CH with GIS, systematic screening of these eight genes has not previously been undertaken.

Objective: Our objective was to evaluate the contribution and molecular spectrum of mutations in eight known causative genes (TG, TPO, DUOX2, DUOXA2, SLC5A5, SLC26A4, IYD, and TSHR) in CH cases with GIS. Patients, Design, and Setting: We screened 49 CH cases with GIS from 34 ethnically diverse families, using next-generation sequencing. Pathogenicity of novel mutations was assessed in silico.

Patients, design, and setting: We screened 49 CH cases with GIS from 34 ethnically diverse families, using next-generation sequencing. Pathogenicity of novel mutations was assessed in silico.

Results: Twenty-nine cases harbored likely disease-causing mutations. Monogenic defects (19 cases) most commonly involved TG (12), TPO (four), DUOX2 (two), and TSHR (one). Ten cases harbored triallelic (digenic) mutations: TG and TPO (one); SLC26A4 and TPO (three), and DUOX2 and TG (six cases). Novel variants overall included 15 TG, six TPO, and three DUOX2 mutations. Genetic basis was not ascertained in 20 patients, including 14 familial cases.

Conclusions: The etiology of CH with GIS remains elusive, with only 59% attributable to mutations in TSHR or known dyshormonogenesis-associated genes in a cohort enriched for familial cases. Biallelic TG or TPO mutations most commonly underlie severe CH. Triallelic defects are frequent, mandating future segregation studies in larger kindreds to assess their contribution to variable phenotype. A high proportion (∼41%) of unsolved or ambiguous cases suggests novel genetic etiologies that remain to be elucidated.

Figure 1.

Schematic illustrating case selection, variant filtering, and distribution of mutations in the cohort of patients studied with CH and GIS. “Solved” cases refers to cases in whom a definitive link was established between genotype and CH phenotype. In “ambiguous” cases, the ascertained genotype could plausibly be contributing to the phenotype, but the evidence to support a causal link was weaker than in the “solved” group, and “unsolved” cases carried no mutations in any of the listed genes. The numbers of cases harboring monoallelic or biallelic mutations in each gene are listed beneath the corresponding gene name for the “solved” cases. Numbers in the intersect between circles denote triallelic cases harboring mutations in both genes. In the “ambiguous” category, the number of mutations in each gene is classified by mutation type beneath the relevant gene name; all except DUOXA2 were monoallelic. “Solved” and “ambiguous” or “unsolved” cases were equally likely to be familial, but CH was generally more severe in the “solved” cases. fs*; frameshift mutation resulting in a premature stop codon; MAF, minor allele frequency; splice; splice region variant, VUS, variant of uncertain significance.

Figure 2.

Summary of TG mutations identified in the study and the associated biochemical phenotype. CH severity is classified according to European Society for Paediatric Endocrinology criteria on the basis of serum fT4 levels; severe, <5, moderate 5 to <10, and mild >10 pmol/liter, respectively (33) and pathogenicity is predicted according to American College of Medical Genetics guidelines (34). A schematic of the TG protein illustrates the position of the mutations relative to the key structural domains of TG including the repetitive type 1, 2, and 3 cysteine-rich regions, acetylcholinesterase homology (ACHE-like) domain and hormonogenic domains. Known mutations are shown in gray, novel mutations in black. *Cases for which complete biochemical data at diagnosis is not available. CH severity refers to sibling. bs, blood spot.

Figure 3.

Summary of TPO mutations identified in the study and the associated biochemical phenotype. CH severity is classified according to European Society for Paediatric Endocrinology criteria (33) and pathogenicity is predicted according to American College of Medical Genetics guidelines (34). The effect of the novel missense mutations was modeled using the phyre2-server. Figures in the top row show the wild-type (WT) model, with amino acids of interest in green; figures on bottom row show the model with the mutant amino acid (orange); local polar contacts are shown with black broken lines. The R291H and R584Q mutations affect amino acids contributing to an intensive network of H-bond contacts close to the catalytic domain involving the heme-group. R291 makes polar contacts with R585 and R582, interacting directly with the heme-group and R584 makes direct polar contacts with the heme-group itself as well as P203 and D633. The mutations R291H (increased hydrophobicity) and R584Q (resulting in a smaller polar group) are likely to disrupt polar contacts affecting local structure and are predicted to affect catalytic activity. The G331V mutation affects local space filling with the larger valine predicted to impair substrate binding by displacement of the nearby helix and/or disruption of polar contacts (orange amino acids, H₂O molecules in blue), affecting the local structure of TPO.

Figure 4.

Summary of DUOX2 mutations identified in the study and the associated biochemical phenotype. CH severity is classified according to European Society for Paediatric Endocrinology criteria (33) and pathogenicity is predicted according to American College of Medical Genetics guidelines (34). Mutation position is illustrated using a schematic representation of the domain structure of the DUOX2 protein. Known mutations are shown in gray and novel mutations in black. The structural model of the peroxidase domain suggests that R354 is part of an intensive hydrogen network. The novel missense mutation R354W replaces the hydrophilic arginine by the hydrophobic tryptophan disrupting this network and also results in a possible repositioning of the loop containing R354 and C351, which mediates interactions between the peroxidase domain and extracellular loops obligatory for DUOX2 function.

Figure 5.

Genotype-phenotype segregation in six kindreds with oligogenic variants. Horizontal bars denote individuals who have been genotyped. Black shading denotes homozygous individuals and half-black shading denotes heterozygotes for TG mutations (F9, F6, F8), TPO mutations (F19, F21), and DUOX2 mutations (F10). Potential oligogenic modulators are included by aligning genotype and phenotype data with the individual to whom they refer in the pedigree. *Cases for whom complete biochemical data at diagnosis are not available (F6b, F8a); CH severity refers to sibling. In F10, black, half-black, and white shading denote the DUOX2 genotype (Q570L homozygous, heterozygous, or wild-type, respectively). The pedigree is annotated with TG genotype in those cases harboring variants (L2547Q, R1691C), and phenotype (euthyroid, transient, or permanent CH) with venous screening TSH results for CH cases. Cases annotated (euthyroid) were born in Pakistan and although euthyroid in adulthood; that they were not screened neonatally for CH may have precluded detection of transient CH.