GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma

Abstract

Mutation detection plays an important role in diagnostic pathology, not only in providing a tissue diagnosis, but also in predicting response to antitumourigenic agents. However, mutation detection strategies are often hampered by masking of mutant alleles by wild-type sequences. Coamplification at lower denaturation temperature PCR (COLD-PCR) reportedly increases the proportion of rare variant sequences in a wild-type background by using PCR cycles in which the denaturation temperature is reduced to favour product formation with lower melt temperatures and heteroduplexes arising from minor variants. Intramuscular myxoma is a rare benign soft tissue neoplasm that occurs sporadically and less commonly in association with fibrous dysplasia (Mazabraud's syndrome). Fibrous dysplasia results from activating GNAS1 mutations, and the same mutations have been identified in small numbers of intramuscular myxoma. The aim of the study was primarily to establish whether COLD-PCR is more sensitive than conventional PCR; this was achieved by testing for GNAS1 mutations in intramuscular myxomas using the two methodologies. Mutations were detected in 8 of 28 (29%) cases of intramuscular myxomas using conventional PCR followed by mutation-specific restriction enzyme digestion (PCR-MSRED) whereas 17 of 28 (61%) mutations were detected using COLD-PCR/MSRED. Mutations were detected in two cases where a diagnosis of low-grade myxofibrosarcoma had been favoured over intramuscular myxoma. No mutations were detected in an additional 9 low-grade and 19 high-grade myxofibrosarcomas, and another 40 control samples. This study shows the power of COLD-PCR compared with conventional PCR in mutation detection, and shows that GNAS1 mutation detection increases diagnostic accuracy when distinguishing between intramuscular myxoma and low-grade myxofibrosarcoma.

A mutation spectrum that includes GNAS, KRAS and TP53 may be shared by mucinous neoplasms of the appendix

“…Somatic activating mutations of GNAS have been reported in several neoplasms, such as pituitary adenomas [20], fibrous dysplasia [21], intramuscular myxomas [22], and villous adenomas of the colon and rectum [23]. Furthermore, GNAS mutations have been reported in low-grade malignancies, including appendiceal tumors, and intraductal papillary neoplasms of the pancreas [24] and bile duct [25], and thus have been considered as characteristic of low-grade tumors.…”

Diagnostic value of investigating GNAS mutations in fibro-osseous lesions: a retrospective study of 91 cases of fibrous dysplasia and 40 other fibro-osseous lesions

“…21,22 This gene is located on chromosome 20q13.3, encoding the a-subunit of the heterotrimeric G (Gsa) protein complex. [23][24][25][26] Two main mutations have been identified within exon 8 of the GNAS gene. 27 These mutations are substitutions in codon 201, leading to the replacement of an arginine by a histidine (R201H), or a cysteine (R201C).…”

“…There are several methods to detect GNAS mutation from paraffin-embedded tissues or frozen samples, ie, Sanger direct sequencing, 39 restriction fragment-length polymorphism from PCR, 11,36,37 an allele-specific probe for PCR quantification, 40 PCR with mutation-specific restriction enzyme digestion, 32 allele-specific PCR, 24 coamplification at lower denaturation temperature PCR, 26 HRM combined with direct sequencing and, more recently, pyrosequencing. 41 The sensitivity of these techniques is variable and GNAS mutations remain unidentified in many fibrous dysplasia lesions, regardless of the molecular method used.…”

<b><i>H-ras</i></b> Mutation Detection in Bladder Cancer by COLD-PCR Analysis and Direct Sequencing

“…COLD-PCR technique is based on the selective PCR amplification of minority alleles from mixtures of wild-type and mutation-containing sequences, regardless of the mutation type or position on the sequence. COLD-PCR is a highly sensitive clinical assay for mutation detection, which could substantially increase mutation-detection sensitivity by 5- to 100-fold with a different downstream detection method, such as direct sequencing and restriction enzyme digestion, in various sample types [17,18,19]. In the current study, COLD-PCR followed by direct sequencing was used to detect H-ras mutations in bladder cancer.…”