A novel missense mutation of the STK11 gene in a Chinese family with Peutz-Jeghers syndrome

The study was approved by the Ethics Committee of Qilu Hospital of Shandong University. The methods were carried out in accordance with the approved guidelines.

Kindred subjects were diagnosed with PJS based on the WHO criteria, and they received polyp surgery in Qilu Hospital of Shandong University. The subject family with PJS participated in this study after providing their informed consent in October 2021. Peripheral blood samples were obtained from all individuals, and comprehensive clinical data, such as medical history, pedigree, physical examination, and endoscopic and pathological findings, were collected. Intestinal polyp specimens were obtained from the proband, her mother, and her grandfather.

Genomic DNA was isolated from peripheral blood of the proband, her brother, and her parents. WES was performed by MyGenostics (Beijing, China) using the Illumina HiSeq X Ten system. The FASTQ files were mapped to the reference human genome (hg19) and then assessed for variant calling using the HaplotypeCaller tool of GATK software.

The potential deleterious effects of the missense mutation in STK11 were predicted using in silico algorithms provided by the online software PolyPhen-2 score (http://​genetics.​bwh.​harvard.​edu/​cgi-bin/​pph2), SIFT (http://​sift.​bii.​a-star.​edu.​sg/​sift-bin/​), MutationTaster (http://​www.​mutationtaster.​org/​cgi-bin/​), and GERP++ (http://​mendel.​stanford.​edu/​SidowLab/​downloads/​gerp/​index.​html). Diagnostic variants were defined as pathogenic or likely pathogenic according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) [14]. Evidence for disease causality was assessed using ClinVar (https://​www.​ncbi.​nlm.​nih.​gov/​clinvar/​). To confirm the conservation of amino acid substitutions in the process of species evolution, the typical protein sequences of multiple different species were aligned using UGENE software to compare mutated positions with conserved domains. Structure prediction was performed by the Swiss-Model online software (https://​swissmodel.​expasy.​org/​). Identified variants were confirmed by Sanger sequencing. The primers used to target human STK11 included forward: 5′-TGCCTGGACTTCTGTGACTTC-3′ and reverse: 5′-CCAGATGTCCACCTTGAAGC-3′.

Four patients distributed across three generations exhibited an autosomal dominant mode of inheritance (Fig. 1A). The age of onset of mucocutaneous pigmentation was as early as one year in II1, and two years in III1 and III2 (Table 1; Fig. 1B, C and D).

I1 had mucocutaneous pigmentation on the lips, face, fingers, toes, and oral mucosa since early childhood. At the age of 15, the patient suffered from intestinal polyps and obstruction and received intestinal surgery at a local hospital. At the age of 50, he presented with a 6-year-history of hematochezia and underwent endoscopy, during which multiple polyps of the colon and rectum were discovered (Fig. 1B). The patient was diagnosed with PJS after pathological examination of the polyps (Fig. 1B) and received surgery for nasopharyngeal cancer in the same year. All resected polyps showed hamartomatous histopathology with peculiar proliferation of smooth muscle cells, growing in a branching-tree pattern and extending into the lamina propria with displacement of the surface epithelium into the submucosa and muscularis propria (Fig. 1B). At the age of 55, he underwent ileocecal resection and ileostomy because of intussusception from polyps. At the age of 57, 19 polyps were resected from the stomach and duodenum. An enhanced computed tomography (CT) examination was performed before surgery, which demonstrated an irregular enhanced mass in the common bile duct with dilation of the bile ducts. A diagnosis of biliary tumors was considered. Magnetic resonance imaging (MRI), magnetic resonance cholangiopancreatography (MRCP), and multiplanar reconstruction (MPR) performed after surgery revealed multiple lamellar long T1 and slightly longer T2 signals in the liver, and a low signal filling defect area in the common bile duct of the liver hilus with a dilated intrahepatic bile duct. A peripheral rim of nodular enhancement was observed in the arterial phase, followed by progressive filling enhancement during the portal venous phase and delayed phase. The upper segment of the common bile duct was thick. The bile ducts were obviously dilated. The walls of the bile duct in the hilar region were thickened, with high signal intensity on diffusion-weighted imaging, and obvious enhancement and delayed enhancement on an enhanced scan (Fig. 1B). He underwent an operation for advanced cholangiocarcinoma (Fig. 1B) later, and died 5 months after surgery.

II1 was a 31-year-old female patient who had been known to have PJS since 2010. The diagnosis was based on familial history of PJS, the presence of characteristic mucocutaneous pigmentation (Table 1), and PJ polyps. She suffered from diffuse abdominal pain and was diagnosed with intussusception by abdominal ultrasonography and CT in 2010. Emergency resection with anastomosis was performed. After that, she underwent endoscopic polypectomy four times from 2012 to 2019, and during this period, she experienced a recurrence of intussusception in December 2019 (Fig. 1C). Resected pedunculated polyps, ranging from 0.3 to 3 cm in diameter, were found in the duodenum, ileocecal region, colon, and rectum (Fig. 1C; Table 1). Postoperative pathology showed characteristics of typical PJ polyps, which was the same as that in her father (Fig. 1C).

III1 was diagnosed with PJS because of familial history of PJS and mucocutaneous pigmentations on the lips, fingers, and oral mucosa, but not on the toes (Table 1). Two polyps were resected from the cavity of the ascending colon and rectum during endoscopic examination. The smaller polyp measured 0.3 × 0.4 cm, and it was covered by red smooth mucosa on the surface; the larger polyp measured 0.5 × 0.6 cm, and it appeared as polypoid hyperplasia with a sessile, rough, and loculated surface. Pathology showed a hamartomatous polyp with normal epithelium and an inflammatory infiltrate with dilated, mucus-filled cystic glands in the lamina propria, consistent with a juvenile polyp (Fig. 1D).

III2 had mucocutaneous pigmentation on the lower lip, but not on the fingers or toes; meanwhile, polyps were not detected in his gastrointestinal tract (Fig. 1D; Table 1).

The proband’s grandmother and aunt were healthy, and they did not have mucocutaneous pigmentation.

To further confirm the diagnosis, we subsequently applied WES in the proband and her family members. A heterozygous missense variant c.521A > C of STK11 was identified in patients III1, III2, II1, and I1 (Fig. 2A). The variant led to the replacement of histidine to proline in codon 174 (p.H174P, Fig. 2B).

Bioinformatic prediction was performed for the mutation, and it yielded a PolyPhen-2 (http://​genetics.​bwh.​harvard.​edu/​pph2/​) score of 1.000, indicating that it was possibly damaging (Fig. 2C), and it also yielded a SIFT (http://​sift-dna.​org/​sift4g) score of 0, indicating that it affected protein function. The amino acid in this residue was evolutionarily conserved (Fig. 2D), which indicated an important functional role. The 3D structures of the mutated protein constructed using the Swiss-model (http://​swissmodel.​expasy.​org) showed loss of the hydrogen bond in the local structure (Fig. 2E) [15]. In addition, we confirmed that this novel mutation was likely pathogenic in these patients with PJS according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) [14], and it broadened the spectrum of STK11 variants associated with PJS. This mutation has not been reported in literatures or recorded in the Human Genome Mutation Database (HGMD, http://​www.​hgmd.​cf.​ac.​uk/​), suggesting that it was a novel variation. The variable site was confirmed and was also identified in her brother, her mother, and her grandfather by Sanger sequencing (Fig. 2A).