Germline Mutation in MUS81 Resulting in Impaired Protein Stability is Associated with Familial Breast and Thyroid Cancer

Abstract

Multiple primary thyroid cancer (TC) and breast cancer (BC) are commonly diagnosed, and the lifetime risk for these cancers is increased in patients with a positive family history of both TC and BC. Although this phenotype is partially explained by TP53 or PTEN mutations, a significant number of patients are negative for these alterations. We judiciously recruited patients diagnosed with BC and/or TC having a family history of these tumors and assessed their whole-exome sequencing. After variant prioritization, we selected MUS81 c.1292G>A (p.R431H) for further investigation. This variant was genotyped in a healthy population and sporadic BC/TC tissues and investigated at the protein level and cellular models. MUS81 c.1292G>A was the most frequent variant (25%) and the strongest candidate due to its function of double-strand break repair. This variant was confirmed in four relatives from two families. MUS81 p.R431H protein exhibited lower expression levels in tumors from patients positive for the germline variant, compared with wild-type BC, and normal breast and thyroid tissues. Using cell line models, we showed that c.1292G>A induced protein instability and affected DNA damage response. We suggest that MUS81 is a novel candidate involved in familial BC/TC based on its low frequency in healthy individuals and proven effect in protein stability.

Keywords: MUS81; breast cancer; exome sequencing; familial cancer; functional analysis; thyroid cancer.

Figure 1

Schematic summary of genes after variant prioritization, including: 17 cancer-related genes with variants, genes with variants carried by families F1 or F2 and by an additional unrelated patient, recurrently altered genes, and genes with recurrent variants. Top bar plot: number of variants by patient/family; Right bar plot: number of variants by gene.

Figure 2

Protein–protein interaction network of 17 cancer related genes and MUS81, highlighting the most likely biologically relevant links among the selected proteins. Nodes represent proteins and lines represent physical protein interactions between them. Red outline identifies the 18 targets, including MUS81 and 17 cancer-related genes, with variants identified by whole-exome sequencing. Gray lines represent low connectivity (low degree), while blue lines link proteins with the highest degree between target genes via XRRC6, SMAD2 and CDK1, and red lines represent direct interactions between targets.

Figure 3

MUS81 c.1292G>A (p.R431H) variant reduces protein stability. (A,B) Breast carcinomas with heterozygous MUS81 variant showed weak protein expression (Score 3). (C)Thyroid carcinomas with heterozygous MUS81 variant showed weak protein immunoreactivity (Score 3). (D) Breast carcinoma wild type for p.R431H had moderate to strong MUS81 expression (Score 6). (E) Normal mammary gland also wild type for MUS81 had elevated protein expression (Score 7). (F). Normal thyroid tissue wild type for the variant had a moderate protein expression (Score 5).

Figure 4

MUS81 c.1292G>A (p.R431H) variant presents reduced stability and reduced DNA damage repair activity. (A,B) Wild-type (WT) or c.1292G>A (p.R431H) MUS81 were ectopically expressed in TPC1 and U87 cell lines. Protein synthesis was blocked with cycloheximide (CHX) and protein decay after one, three, and six hours was evaluated by Western blot. (A) Representative Western; (B) quantification of MUS81 levels relative to CHX untreated cells (mean of three independent experiments ± standard error of the mean (SEM)). Two-way ANOVA followed by Dunnett’s post hoc test (WT vs. MT * p < 0.05). (C). TPC1 cells were transfected with empty vector, MUS81 or MUS81 p.R431H and treated or not with cisplatin for 1 h. Cells were fixed and labeled for phosphorylated Histone H2AX. (D). The percentage of yH2AX labeled cells was quantified. Two-way ANOVA followed by Dunnett’s post hoc test (control: Empty vector vs. MUS81 WT p > 0.05, Empty vector vs. MUS81 p.R431H * p < 0.05; MUS81 WT vs. MUS81 p.R431H * p < 0.05; Cisplatin 10 μM: Empty vector vs. MUS81 WT * p < 0.05, Empty vector vs. MUS81 p.R431H p > 0.05; MUS81 WT vs. MUS81 p.R431H p > 0.05).