Identification of a Cowden syndrome patient with a novel PTEN mutation and establishment of patient-derived induced pluripotent stem cells

Abstract

Cowden syndrome (CS) is an autosomal dominant inherited disorder characterized by multiple hamartomas in various organs such as the mucosa, skin, and gastrointestinal tract. Patients with CS are at high risk for breast and thyroid cancers. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that negatively regulates the AKT pathway, and PTEN mutations are known to be the major causes of this syndrome. However, the pathogenesis of this syndrome has not been clarified. Here, we present a case of a Japanese woman with multiple oral polyps, breast cancer, and thyroid cancer who was clinically diagnosed with CS. We obtained DNA and RNA samples from the patient's peripheral blood mononuclear cells (PBMCs) and buccal mucosa tumor. Next-generation sequencing revealed novel germline mutations (c.1020delT and c.1026G > A) in exon 8 of PTEN. Sanger sequencing identified no PTEN transcript from the mutant allele. Furthermore, CS-specific induced pluripotent stem cells (CS-iPSCs) were established from PBMCs of the patient under feeder- and serum-free culture. Compared with healthy PBMCs and iPSCs, both of the CS-derived PBMCs and CS-iPSCs exhibited significantly reduced expression of the PTEN transcript. The transcriptional variant, PTENδ, was increased in CS-iPSCs, suggesting that it may be the cause of the disease.

Keywords: Cowden syndrome; Feeder- and serum-free culture condition; Induced pluripotent stem cells; Phosphatase and tensin homolog deleted on chromosome 10; Phosphatase and tensin homolog deleted on chromosome 10 δ.

Fig. 1

Genetic analysis (A, B). Mutation of PTEN identified by next-generation sequencing (NGS) (A) and Sanger sequencing (B).

Fig. 2

Characterization of the CS-iPSC. (A) Alkaline phosphatase staining. (B) Induction efficiency of CS-PBMC was higher than that of WT-PBMCs. (C) Phase contrast. (D, E, F, H, I) IF and RT-PCR assay for expression of hES cell surface markers. Each bar indicates 100 μm in length. (G) In vivo teratoma formation assay. Neural tube epithelium (left) and intestinal epithelium (middle) (H&E stained) and cartilage (right) (Alcian Blue-PAS-Stain). Each bar indicates 100 μm in length.

Fig. 3

PTEN gene expression and PTEN/AKT pathway activity. (A, B) PTEN mRNA expression in CS-PBMC and -iPSC. (C) PTEN mRNA sequencing by the Sanger method. (D) PTEN and AKT protein expression in CS-iPSC. (E) Analysis of AKT signaling activity by the Bio-Plex assay.

Fig. 3

PTEN gene expression and PTEN/AKT pathway activity. (A, B) PTEN mRNA expression in CS-PBMC and -iPSC. (C) PTEN mRNA sequencing by the Sanger method. (D) PTEN and AKT protein expression in CS-iPSC. (E) Analysis of AKT signaling activity by the Bio-Plex assay.

Fig. 4

PTEN δ expression in iPSCs. (A) PTEN δ mRNA expression in CS-iPSC was higher than that in WT-iPSCs. (B) PTEN δ sequencing from iPSCs by the Sanger method. (C) Predicted protein sequence variant of these mutations.