Identification of a KEAP1 germline mutation in a family with multinodular goitre

Abstract

Background: The familial clustering of multinodular goitres (MNGs) with a dominant mode of inheritance has been repeatedly reported. Linkage studies have revealed several genetic loci responsible for familial MNG; however, most of the causative variants remain unknown.

Methods and results: Through linkage analysis using single-nucleotide polymorphism markers, we identified a new MNG locus on 19p13.2-q12 in a five-generation Japanese MNG family. Subsequent mutation searches focusing on the candidate 25-Mb region of chromosome 19 identified a heterozygous mutation, c.879_880delinsA, p.Asp294Thr, fs*23, in exon 3 of the KEAP1, which plays a central role in the cytoprotection pathway against oxidative stress. Reverse transcriptase-PCR analysis showed low expression of wild type KEAP1 accompanied by no transcription product of mutant allele in the normal and goitre region of thyroid tissues obtained from the proband. In agreement with previous studies showing that KEAP1 negatively regulates NFE2L2, the NFE2L2 target genes GSTA4 and GCLC were up-regulated in the thyroid tissues of the patient.

Conclusions: This study identified the first KEAP1 mutation in MNG. The results provide insights into the pathogenesis of goitre which develops in the organ continuously exposed to oxidative stress during hormone synthesis.

Figure 1. A pedigree of a five-generation multinodular goitre family.

Individuals are numbered above each symbol. Individuals affected with MNG are indicated by filled symbols and age of onset is described under the patient symbol. The arrow shows a proband of this family study. The bars above each symbol indicate individuals included in the linkage analysis.

Figure 2. Summary of the genome-wide SNP linkage analysis.

(A) The multipoint parametric LOD (pLOD) score with a marker interval of 1.0 cM is shown for all of the autosomes and the X chromosome. (B) The multipoint pLOD and the information score of chromosome 19 are shown. The black and red lines (or dot) denote results from 1.0 and 0.2 cM marker interval analysis, respectively.

Figure 3. Identification of a heterozygous mutation in KEAP1 .

(A) The result of Sanger sequencing of proband DNA showed the c.879_880delinsA mutation (red), resulting in a 1-base deletion and a frameshift (p.Asp294Thr, fs*23) in KEAP1. (B) Domain structure and mutation location in the KEAP1 protein. The protein consists of an N-terminal region (NTR; amino acids 1 to 60), a BTB domain (amino acids 61 to 179), an intervening region (IVR; amino acids 180 to 314) and a DC domain (amino acids 315 to 624). The BTB and N-terminal portion of IVR is responsible for dimerisation and the interaction with CUL3. The DC domain is also critical for the interaction with NFE2L2. The p.Asp294Thr, fs*23 mutation is located in the IVR. The reported frequencies of the somatic mutations observed in each domain in cancer cells are shown at the bottom .

Figure 4. Reverse transcriptase-PCR for mutant and wild type KEAP1 .

(A) Primer positions for reverse transcriptase-PCR in KEAP1 exons. The asterisk indicates the position of c.879_880delinsA mutation in exon 3. The primer, KEAP1-mut-rvs, contains "T" nucleotide at the 3' end which is the complement of mutant "A" nucleotide to amplify mutant allele. The expected sizes of PCR products are as follows: 233 bp for KEAP1-N-fwd and KEAP1-N-rvs; 201 bp for KEAP1-C-fwd and KEAP1-C-rvs; 282 bp for KEAP1-mut-fwd and KEAP1-mut-rvs. (B) Results of the reverse transcriptase-PCR for the mutant and wild type KEAP1. cDNAs from control normal thyroid, the normal and goitre region of thyroid tissues obtained from the proband were amplified by 3 primer sets indicated in (A) to mutant KEAP1 (upper panel), 5' and 3' normal portions adjacent mutation site (second and third panels, respectively) of KEAP1. GAPDH was amplified as a control for the amount of cDNA in each sample. The expected size of PCR product of GAPDH is 452 bp. In the experiment of mutant KEAP1 amplification, the plasmids containing mutant or wild type KEAP1 (pCMV-myc-KEAP1mut or pCMV-myc-KEAP1wt, respectively) were utilized as control templates.

Figure 5. Up-regulation of NFE2L2 target genes in the thyroid tissues of the patient.

Results of the quantitative PCR of NFE2L2 and its target genes GSTA4, GCLC and NQO1 in the normal and goitre region of thyroid tissues obtained from the proband. The expression levels relative to control RNA from an adult thyroid are shown. The significance with the t-test is indicated with asterisks (*p<0.05 and **p<0.01).