Nodule-Specific NRF2-Targeted Upregulation in Patients With KEAP1 Mutations and Familial Nontoxic Multinodular Goiter

Abstract

Context: Kelch-like ECH-associated protein 1 (KEAP1) is associated with nuclear factor erythroid-2-related factor 2 (NRF2) and promotes NRF2 degradation in normal conditions. Genetic abnormality in KEAP1 is a rare disease and presents with familial multinodular goiter.

Objective: This study assessed the clinical and molecular findings concerning nodular formation in the thyroid gland of patients harboring KEAP1 germline mutations.

Methods: Next-generation sequencing analysis targeting goiter-associated genes was performed on 39 patients with familial multinodular goiter. The expression of NRF2-targeted genes from surgical thyroid specimens of patients with KEAP1 mutations were analyzed using a whole-transcript expression array and immunohistochemistry.

Results: We found 5 probands with pathogenic heterozygous mutations in KEAP1 (p.Q86*, p.L136P, p.V411fs, p.R415C, and p.R483H) that had no meaningful concomitance with mutations of other goiter-associated genes at germline and somatic levels. Their common histopathological features showed multinodular goiters in the entire thyroid gland with few degenerative lesions or complications of malignancy and slow proliferation indicating less than 1% at the Ki-67 labeling index. Among 42 NRF2-targeted genes, antioxidant genes were most frequently upregulated (11/12) in the nodule, followed by detoxification genes (6/11). Immunohistochemical analysis showed relatively high expression of glutathione peroxidase 2 and NAD(P)H quinone oxidoreductase 1 (representative NRF2-targeted genes) in the nodules of various patients harboring KEAP1 mutations.

Conclusion: KEAP1 germline heterozygous mutations exert excessive NRF2 activity in the thyroid gland and may confer cytoprotective effects even under abundant reactive oxygen species associated with thyroid hormone production, resulting in thyroid hyperplasia with scarce degradation.

Keywords: KEAP1 germline mutation; NRF2-targeted gene; familial goiter; multinodular goiter.

Figure 1.

Flowchart of the study population. Abbreviations: FFPE, formalin-fixed paraffin-embedded; MNG, multinodular goiter.

Figure 2.

Identifying families with Kelch-like ECH-associated protein 1 (KEAP1) mutations. (A) Localization of germline KEAP1 mutations. The structure is represented by functional regions and corresponding amino acid numbers. Five mutations were identified in our hospital and 1 mutation was reported in another institute. (B) Pedigree of the investigated family. Individuals affected by goiter are indicated by filled symbols. The arrow shows the proband. The bars above each symbol indicate individuals who underwent sequencing analysis for KEAP1.

Figure 3.

(A-D) Histopathological features and the (E) and (F) Ki-67 labeling index in the thyroid nodules of 4 probands with Kelch-like ECH-associated protein 1 (KEAP1) mutations.

Figure 4.

Scatter plots of RNA expression levels. Each gene expression is distributed at the intersection of the thyroid nodular lesion (vertical axis) and normal parenchyma (horizontal axis) of the patient with a Kelch-like ECH-associated protein 1 (KEAP1) mutation (L136P). The expression of GPX2, NQO1, and TG is indicated in the blue circle.

Figure 5.

Immunohistochemical detection of NRF2 target genes in the thyroid. (A-D) The upper panels represent a patient with a Kelch-like ECH-associated protein 1 (KEAP1) mutation (L136P), and (E-H) the lower panels represent a control patient harboring no KEAP1 mutation. Histopathological feature of nonnodular parenchyma (*) and thyroid nodules (#) in A and E, hematoxylin-eosin staining; B and F, TG; C and G, NQO1; and D and H, GPX2 expression. The data are representative of 2 experiments with similar results.

Figure 6.

Kelch-like ECH-associated protein 1 (KEAP1) mutations and nuclear factor erythroid-2 related–factor 2 (NRF2)-targeted gene expression. (A-D) TG; (E-H) NQO1; and I to L, GPX2 expression in the thyroid of 4 probands with different KEAP1 mutations. *Nonnodular parenchyma; ^#Thyroid nodules.