Whole-exome sequencing studies of nonhereditary (sporadic) parathyroid adenomas

Abstract

Context: Genetic abnormalities, such as those of multiple endocrine neoplasia type 1 (MEN1) and Cyclin D1 (CCND1) genes, occur in <50% of nonhereditary (sporadic) parathyroid adenomas.

Objective: To identify genetic abnormalities in nonhereditary parathyroid adenomas by whole-exome sequence analysis.

Design: Whole-exome sequence analysis was performed on parathyroid adenomas and leukocyte DNA samples from 16 postmenopausal women without a family history of parathyroid tumors or MEN1 and in whom primary hyperparathyroidism due to single-gland disease was cured by surgery. Somatic variants confirmed in this discovery set were assessed in 24 other parathyroid adenomas.

Results: Over 90% of targeted exons were captured and represented by more than 10 base reads. Analysis identified 212 somatic variants (median eight per tumor; range, 2-110), with the majority being heterozygous nonsynonymous single-nucleotide variants that predicted missense amino acid substitutions. Somatic MEN1 mutations occurred in six of 16 (∼35%) parathyroid adenomas, in association with loss of heterozygosity on chromosome 11. However, no other gene was mutated in more than one tumor. Mutations in several genes that may represent low-frequency driver mutations were identified, including a protection of telomeres 1 (POT1) mutation that resulted in exon skipping and disruption to the single-stranded DNA-binding domain, which may contribute to increased genomic instability and the observed high mutation rate in one tumor.

Conclusions: Parathyroid adenomas typically harbor few somatic variants, consistent with their low proliferation rates. MEN1 mutation represents the major driver in sporadic parathyroid tumorigenesis although multiple low-frequency driver mutations likely account for tumors not harboring somatic MEN1 mutations.

Fig. 1.

Details of somatic variants in the discovery set of 16 parathyroid adenomas. A, The number of somatic variants identified in each of the 16 parathyroid adenomas is shown (Table 1). A total of 212 somatic variants were observed, and more than 50% of these occurred in tumor 6. Tumors had a median of eight variants per sample (range, 2–110), and those harboring somatic MEN1 mutations are identified with an asterisk. B–D, The somatic variants occurring in 15 parathyroid adenomas (black bars) with an apparent low somatic mutation rate (total variants = 102) are shown separately from those that occurred in tumor 6 (total variants = 110) (gray bars). B, Chromosomal distribution. The variants per megabase (Mb) of exonic DNA are shown, thereby correcting for the size of each chromosome. Overall, the mean (±sd) somatic mutation rate in the 15 tumors was 0.17/Mb (±0.08) of exonic DNA, whereas it was >30 sd higher at 2.8/Mb exonic DNA in tumor 6. The variants were distributed across chromosomes in the set of 15 tumors and tumor 6, although the number of variants per chromosome was small such that it did not allow statistical significance to be established (χ² P = 0.62 for the 15 tumors, P = 0.57 for tumor 6). C, SNV types. Missense SNVs represented the most common type of variant in all tumors. However, insertions or deletions (indels) contributed approximately 13% of variants in the 15 tumors, whereas only about 2% of those occurring in tumor 6. D, Nucleotide alterations. Transitions were more common than transversions in all tumors. However, C:G→T:A transitions were the most frequently (51%) observed SNV in the 15 tumors, whereas tumor 6 had a greater proportion (58%) of A:T→G:C transitions. FS, Frameshift.

Fig. 2.

Chromosomal LOH in parathyroid tumors 5 and 6. Exome sequence data allowed coarse LOH mapping to detect large areas of chromosomal loss for each parathyroid adenoma. Areas of chromosomal loss within the tumor sample (blue line) are indicated by an increased homozygous variant fraction across the chromosome when compared with the paired leukocyte DNA sample (green line). A, Example of LOH mapping for tumor 5 containing a somatic MEN1 mutation (Table 2). This demonstrates likely areas of chromosomal loss on chromosome 11 (MEN1 locus) as well as chromosomes 15 and 18 as indicated by the increased homozygous variant fraction (blue line) in the tumor sample compared with the paired leukocyte sample (green line). All tumors harboring somatic MEN1 mutations demonstrated LOH affecting chromosome 11 (Table 1) in keeping with biallelic inactivation of MEN1. B, LOH mapping for tumor 6 harboring the POT1 (located on chromosome 7) mutation demonstrates chromosomal loss affecting the majority of chromosome pairs (17 of 23), indicating widespread LOH and in keeping with increased genomic instability. The chromosome number (1–22 and X) is shown within each panel; the x-axis represents chromosomal location (scale; base position on chromosome × 10⁸), and the y-axis represents homozygous variant fraction.

Fig. 3.

Studies of POT1 mutation identified in tumor 6. A, DNA sequence analysis of leukocyte (L) and tumor (T) DNA from patient 6 (Table 1) confirmed the occurrence of a hemizygous POT1 somatic mutation (m), c.G546C, in the tumor with LOH of chromosome 7 and its absence in the wild-type (WT) leukocyte. The G to C transition affected the last nucleotide of exon 8. B, The mutation predicted a splicing defect that would result in skipping of exon 8, which is 291 bp in size, and this was confirmed using RNA from the tumor of patient 6 and RT-PCR that used POT1-specific primers for exons 6 and 7 (6/7F) and for exons 9 and 10 (9/10R). Thus, RT-PCR of control leukocyte RNA (n = 3) (one shown: Control, L) and control parathyroid adenomas (n = 3) (one shown: Control T), which did not have the POT1 c.G546C mutation, yielded products of 599 bp in size, whereas those from tumor 6 (Patient 6, T) were of 308 bp, consistent with a loss of 291 bp, which is the size of exon 8. Control PCR using reverse transcriptase-negative samples (−) resulted in the absence of products, thereby demonstrating the specificity of the RT-PCR. C, DNA sequence analysis of the RT-PCR products from tumor 6 (m) and WT control (one shown) samples revealed normal splicing of exon 7 to exon 8 in the WT but abnormal splicing of exon 7 to exon 9 in tumor 6, consistent with skipping of exon 8. D, Loss of exon 8 is predicted to result in an in-frame deletion of 98 amino acids of the protection of telomeres 1 (POT1) protein (p.Lys85_Val183del). WT POT1 has two conserved OB fold domains (OB1, residues 9–141, and OB2, residues 161–278) that are responsible for single-stranded DNA binding and telomere protection. Exon 8 encodes 98 amino acids of the POT1 protein and its loss is predicted to result in an in-frame loss of 98 amino acids (p.Lys85_Val183del), which likely results in loss of DNA-binding capacity and hence loss of function.