Recurrent Hyperparathyroidism Due to a Novel CDC73 Splice Mutation

Presentation Number: FRI 332
Date of Presentation: April 1st, 2016

Namita G. Hattangady*, Tremika Le-Shan Wilson, Barbra S. Miller, Antonio M Lerario, Thomas J Giordano, Palak Choksi and Tobias Else
University of Michigan, Ann Arbor, MI

Abstract

Background: Primary hyperparathyroidism (pHPT) is commonly caused by adenomas, less frequently by multiglandular parathyroid hyperplasia and rarely by parathyroid cancer. pHPT is mostly sporadic but occurs as part of familial syndromes such or multiple endocrine neoplasia type 1 (MEN1) or Hyperparathyroidism-Jaw Tumor (HPT-JT) syndrome. HPT-JT is caused by inactivating mutations in the tumor suppressor gene CDC73 (or HPRT2, which codes for the protein parafibromin). Herein we investigate an intronic germline mutation in CDC73 in a patient with recurrent pHPT despite multiple surgeries.

Clinical case: The index case is a 52 year old male with a long standing history of kidney stones who was diagnosed with pHPT at age 35. The initial surgery removed two hyperplastic parathyroid glands resulting in normalization of calcium levels. Seven years later, the patient had recurrent pHPT and underwent removal of two adenomatous glands and autotransplantation to the forearm. Since then he had recurrence of pHPT and unsuccessful surgery to reduce parathyroid tissue in the forearm. The probands’s family history was negative for pHPT, jaw tumors or kidney lesions. The proband’s mother, however, did undergo a hysterectomy, possibly due to uterine fibroids. Initial genetic testing did not reveal a mutation in MENIN, but revealed the presence of a germline intronic variant of uncertain significance (VUS) in CDC73c.238-8G>A (IVS2-8G>A).

Methods: In order to gather in vitro evidence for pathogenicity of the mutation, we analyzed loss of heterozygosity and gene splicing as the mutation was predicted to activate a cryptic splice site, resulting in early translation termination. Genomic DNA from formalin fixed paraffin embedded parathyroid tumor tissues showed loss of heterozygosity, while it was preserved in control thyroid tissue. Immunostaining for parafibromin confirmed protein expression in thyroid tissue and loss of expression in the parathyroid adenoma tissues. With regards to an effect on splicing, both predicted transcripts were found by RT-PCR in mRNA from peripheral blood. In vitro characterization of the splice variant was confirmed by cloning the genetic fragment containing the wildtype and mutant intronic region and its successive exon into the pDUP4-1 minigene vector and expressing it in human embryonic kidney (HEK293 cells) by transfection. Cells expressing the pDUP4-1/Mutant CDC73 displayed a splice variant of CDC73 which was 6 base pairs longer than the wild type CDC73 mRNA.

Conclusion: In summary, we show that this novel intronic CDC73 mutation, c.238-8G>A, activates a cryptic splice site and that the wild type allele is lost in this patient’s parathyroid adenomas. Together, these findings provide in vitro evidence to reclassify this mutation as a pathogenic variant confirming our patient’s diagnosis of CDC73-related disorder and an increased risk for parathyroid cancer development.

 

Nothing to Disclose: NGH, TLSW, BSM, AML, TJG, PC, TE