Genotype of CDC73 germline mutation determines risk of parathyroid cancer

in Endocrine-Related Cancer

Correspondence should be addressed to Y Li or H Chen or W F Simond: yulongli@pennstatehealth.psu.edu or haobin.chen@nih.gov or bills@niddk.nih.gov
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Mutation of the CDC73 gene, which encodes parafibromin, has been linked with parathyroid cancer. However, no correlation between genotypes of germline CDC73 mutations and the risk of parathyroid cancer has been known. In this study, subjects with germline CDC73 mutations were identified from the participants of two clinical protocols at National Institutes of Health (Discovery Cohort) and from the literature (Validation Cohort). The relative risk of developing parathyroid cancer was analyzed as a function of CDC73 genotype, and the impact of representative mutations on structure of parafibromin was compared between genotype groups. A total of 419 subjects, 68 in Discovery Cohort and 351 in Validation Cohort, were included. In both cohorts, percentages of CDC73 germline mutations that predicted significant conformational disruption or loss of expression of parafibromin (referred as ‘high-impact mutations’) were significantly higher among the subjects with parathyroid cancers compared to all other subjects. The Kaplan–Meier analysis showed that high-impact mutations were associated with a 6.6-fold higher risk of parathyroid carcinoma compared to low-impact mutations, despite a similar risk of developing primary hyperparathyroidism between two groups. Disruption of the C-terminal domain (CTD) of parafibromin is directly involved in predisposition to parathyroid carcinoma, since only the mutations impacting this domain were associated with an increased risk of parathyroid carcinoma. Structural analysis revealed that a conserved surface structure in the CTD is universally disrupted by the mutations affecting this domain. In conclusion, high-impact germline CDC73 mutations were found to increase risk of parathyroid carcinoma by disrupting the CTD of parafibromin.

Supplementary Materials

    • Supplementary Figure 1. Comparison of clinical features in the subjects with parathyroid classic adenomas, atypical parathyroid adenomas, or parathyroid carcinomas. The dot plots show difference between the marked groups on a) age at the diagnosis of hyperparathyroidism; b) parathyroid tumor size; c) serum calcium levels; and d) serum PTH levels.
    • Supplementary Figure 2. Kaplan-Meier plots of event-free survival of parathyroid carcinoma (left) and hyperparathyroidism (right) in several subsets of the subjects grouped by high-or low-impact germline mutationsof the CDC73 gene: (a,b) Subjects with CDC73germline mutations that were confirmed by DNA sequencing; (c,d) subjects who were index cases; and (e,f) subjects after atypical parathyroid adenomas were excluded.
    • Supplementary Figure 3. Kaplan-Meier plots of event-free survival of parathyroid carcinoma (a) and hyperparathyroidism (b) in the subjects grouped by mutation types of high-impact mutations in comparison to the subjects with low-impact mutations.
    • Supplementary Figure 4. Impact of the mutations on expression of parafibromin and binding to CTR9 (a key component of PAF1 complex). a) Various Flag-tagged CDC73 (HRPT2) mutants and WT were transiently expressed in HEK 293 cells and expression of parafibromin was examined by western blot using an anti-Flag antibody at 48 hours after transfection. b) Co-immunoprecipitation of HA-tagged CTR9 and Flag-tagged CDC73 WT/mutants using anti-HA antibody. Rabbit IgG isotype control (Cell Signaling) was used as a negative control for IP (IgG IP). The presence of Flag-tagged CDC73 WT/mutants and HA-tagged CTR9 in the IP eluates were detected by western blot using anti-Flag HRP-conjugated antibody and anti-HA HRP-conjugated antibody, respectively. WT CDC73 was used as a positive control and the c.406A>T (p.136X) mutant, which lacks the central CTR9 binding sequence (aa. 226-413), was used as a negative control. The low-impact mutations are highlighted in light green color and high-impact mutations are in magenta color; the parathyroid carcinoma-associated mutations are underlined. The experiments were performed at least twice.
    • Supplementary Figure 5. Kaplan-Meier plots of event-free survival of parathyroid carcinoma (left) and hyperparathyroidism (right) in several subsets of the subjects grouped by the mutations affecting the NTD only (N), the CTD only (C) or both protein domains (N+C): (a,b) Subjects with sequencing-confirmed CDC73germline mutations; (c,d) subjects who were index cases; and (e,f) subjects after atypical parathyroid adenomas were excluded.
    • Supplementary Table 1. List of the published case reports/case series included in the present study.
    • Supplementary Table 2. Mutation type, genotype group and parathyroid phenotype of all subjects included in the present study.
    • Table S3. The percentages of the low-impact and the high-impact mutations in the subjects who developed jaw disease, kidney disease or uterine disease. The ratio of the above mutations in the subjects who developed the specified disease was compared to the ratio in all the subjects. The significance of the difference was calculated using chi-square or Fisher’s exact test, as appropriate.
    • Table S4. The percentages of the mutations impacting on the NTD only, the CTD only, or both domains in the subjects with jaw disease, kidney disease, or uterine disease. The ratios of the above mutations in the subjects who developed the specified disease were compared to the ratio in all the subjects. The significance of the difference was calculated using chi-square or Fisher’s exact test, as appropriate.

 

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