Identification a nonsense mutation of APC gene in Chinese patients with familial adenomatous polyposis
- Authors:
- Published online on: February 14, 2017 https://doi.org/10.3892/etm.2017.4122
- Pages: 1495-1499
Abstract
Introduction
Familial adenomatous polyposis (FAP; MIM 175100) is a rare autosomal dominant disorder, which is characterized by the development of numerous adenomatous polyps throughout the colon and rectum (1). It is a pre-cancerous disease, which develops into colorectal cancer (CRC) in almost all patients without early diagnosis and colorectal surgery (2). FAP may have extracolonic manifestations, including osteomas, dental abnormalities, congenital hypertrophy of the retinal pigment epithelium (CHRPE) and upper gastrointestinal polyps (3). The incidence of FAP at birth is estimated to be 3–10 per 100,000 individuals (4).
FAP has three phenotypes: Classic FAP (CFAP), attenuated FAP (AFAP) and MUTYH-associated polyposis (MAP) (5), with CFAP and AFAP being autosomal dominant disorders. It has been identified that the adenomatous polyposis coli (APC) gene on chromosome 5q22.2 is associated with CFAP and AFAP (6). MAP is a recessive dominant disorder caused by mutations in the MUTYH gene (7). In the present study, mutations of the APC gene were detected in a Chinese family with CFAP by sequencing analysis, and a nonsense mutation was identified.
Materials and methods
Patients
A 40 year-old Chinese male patient was seen at the Department of Emergency of the Second People's Hospital of Heifei (Hefei, China) in August 2011, due to experiencing hematochezia for 1 day. In the past year, he had frequently suffered from moderate diarrhea with scurrying pain around the umbilicus. His medical history was not indicative of colitis and hemorrhoids. The patient was a non-smoker and drank alcohol socially. Colonoscopy findings revealed a huge neoplasm with surface erosion and bleeding 35–38 cm away from the anus (Fig. 1A), as well as congestion, edema and diffused polyps with diameters of 0.3–0.8 cm from the ascending colon to the rectum mucosa (Fig. 1B). The primary diagnosis was FAP. Subsequently, the patient successfully underwent laparoscopic total colectomy and ileal anal anastomosis. Postoperative recovery was good. Pathological findings revealed an abundance of multiple tubular papillary adenoma with low-level intraepithelial neoplasia throughout the entire colon. The family comprised 18 members that spanned three generations, including 3 male (one of which was the proband of the present study) and 2 female individuals affected by FAP (Fig. 2). A similar disease course and abnormalities were found in these patients.
Mutational analysis
The protocol of the present study was approved by the Ethics Committee of the Second People's Hospital of Heifei (Hefei, China) and Zhongshan Hospital (Shanghai, China) and all patients provided written informed consent to be included in the present study. Peripheral blood samples were obtained from the four living patients (II:3, II:6, II:8 and the proband III:2; Fig. 2). In addition, samples from 100 unrelated population-matched controls from Zhongshan Hospital were sequenced for mutations to exclude the possibility that it is a polymorphism in the APC gene. DNA was extracted according to standard methods. Primers flanking all 15 coding exons and intron-exon boundaries of the APC gene were extracted using the web-based version of the Primer 3.0 program (http://primer3.ut.ee/). The primers used are listed in Table I. The APC gene of this family was analyzed by direct sequencing in reaction conditions as previously described (8). Subsequent to amplification, a QIAquick PCR Purification kit (Qiagen, Hilden, Germany) was used to purify the products. The APC gene was sequenced using an ABI PRISM® 3730 automated sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sequence comparisons and analysis were performed using Phred-Phrap-Consed version 12.0 software (http://www.phrap.org/phredphrapconsed.html). Mutations were identified by comparison with the reported complementary DNA reference sequence (GenBank accession no, NM_000038).
Results
Sequencing results of the proband revealed a nonsense mutation (c.2971G>T, p.Glu991*) located at exon 16 of the APC gene (Fig. 3A). This mutation was also verified in the other three patients, but excluded in the unaffected family members and 100 unrelated population-match controls (Fig. 3B). This mutation forms a premature stop codon at amino acid residue 991, which results in a truncated protein short of 1,853 amino acids. This mutation has already been reported a patient with hereditary cancer-predisposing syndrome (https://www.ncbi. nlm.nih.gov/clinvar/15587313/). The present study confirmed this mutation in a Chinese family with CFAP. The result demonstrates that this mutation may be a hotspot mutation in diverse population.
Discussion
FAP is an autosomal dominant disease characterized by the development of hundreds to thousands of adenomas in the colon and rectum, and is at times accompanied with certain extra-intestinal manifestations such as CHRPE, dental disorders and desmoid tumors (9). APC is a tumor suppressor gene located on the long arm of chromosome 5 in band q21, whose mutation is responsible for CFAP and AFAP. The length of the gene is 108,353 bp and it is divided into 15 exons (10). The APC protein has multiple domains that mediate oligomerization as well as binding to a variety of intracellular proteins and has a central role in Wnt signaling by regulating of degradation of proteins associated with this pathway (11).
To date, according to the information available in public databases, such as The Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php), >1,000 different APC mutations have been reported, among which >100 cases were contributed by Chinese studies (12–17). While the type of APC gene mutation varies, nonsense and frameshift mutations are most frequently seen, and have been predicted to produce truncated proteins, finally leading to the development of diseases (17).
According to certain studies, most FAP patients inherit one APC allele mutation from their parents with the other allele being normal. Diseases would not occur until the normal allele undergoes a new mutation (11). It has also been estimated that new germline mutations of APC account for one third of FAP patients who have no family history of FAP (18). Certain studies have attempted to explore the correlation between specific APC mutations with the clinical phenotype. Certain correlations do exist, for instance, mutations between codons 169 and 1,578 were generally associated with CFAP (19–21). Mutations downstream of codon 1,596 are frequently seen in AFAP (11). Mutations between codons 1,445 and 1,578 were associated with desmoid tumors, whereas those between codons 279 and 1,309 were correlated with the development of duodenal polyposis (22–24). While it appears promising to predict a patient's phenotype by the mutation site of the APC gene, this was proven to not be feasible in clinical practice. Considerable variability has been found in the presentation of specific phenotypes in patients with identical mutations (25). This indicates that the phenotype is associated with more factors than genetic mutations (25).
In the present study, the nonsense mutation c.2971G>T (p.E991*) was identified in exon 15 of the APC gene. The resulting truncated protein lacked 1,853 amino acids. The wild-type sequence in the affected region of the APC gene is highly evolutionarily conserved in different species, including humans, mice, rats, frogs, zebrafish and pufferfish. Through mutation-associated truncation, the APC protein loses its microtubule binding domain, end binding-1 binding domain, β-catenin degradation domain and β-catenin binding domain, which is likely to affect the proliferation and differentiation status of cells and eventually results in colorectal polyps and cancer (26,27). In addition, this nonsense mutation may lead to nonsense-mediated decay of APC transcripts. The mutation results in haplo-insufficiency of APC, which leads to development of diseases.
While evidence strongly links APC gene mutations with FAP, the single factor is not sufficient to explain the etiology of the disease. It is estimated that 10–30 percent of patients with classical FAP do not have any detectable APC mutation. A proportion of FAP patients have MAP, an autosomal recessive polyposis syndrome caused by biallelic mutations in the MUTYH gene. Therefore, it is recommended that patients who have a recessive family history of FAP are evaluated for a MUTYH mutation (28).
Surgery remains to be the only option to cure the disease, although it remains debatable which surgical option is the golden standard. However, given the substantial risk of rectal cancer developing after colectomy and ileorectal anastomosis, most experts recommend total proctocolectomy for typical FAP patients with multiple rectal adenomas (29). Diet and drugs have been shown to have a role in preventing cancer. Caloric restriction or diet with olive oil, fruits and vegetables significantly reduced the number of polyps in a mouse model of multiple intestinal neoplasia with genetically manipulated APC (30). Randomized trials have shown that celecoxib causes regression of established adenomatous polyps in individuals with FAP. In 2001, the US Food and Drug Administration approved the use of celecoxib in patients with FAP presenting with polyps (31). The proband of the present study successfully underwent laparoscopic total colectomy and ileal anal anastomosis and postoperative recovery was good.
In conclusion, the present study identified a mutation in the APC gene in a Chinese family with FAP. The present study added novel variants to the knowledge of APC mutations in FAP. Identification of novel mutations will be useful to reveal the correlation between genotypes and phenotypes.
Acknowledgements
This study was supported by a grant from the Shanghai Science and Technology Innovation Action Plan (nano-science and technology projects; no. 12nm0501402). The authors would like to thank all patients and control individuals for their participation in this study.
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