Comprehensive analysis of the genetic mutation spectrum of Koreans being evaluated for familial adenomatous polyposis (FAP)

Abstract

Familial adenomatous polyposis (FAP) is a cancer predisposition syndrome inherited in an autosomal dominant pattern. The presence of hundreds to thousands of colorectal adenomas in the colon is a characteristic manifestation of FAP, with various extracolonic manifestations. Mutations in the adenomatous polyposis coli (APC) gene are known to be associated with FAP. The majority of the APC gene mutations are frameshift or nonsense mutations leading to a truncation of the APC protein. As a known tumor suppressor gene, a deleterious gene produced by inactivating germline mutations in the APC gene is unable to suppress cellular growth and lead to formation of adenomas, which can subsequently show malignant potential. Characterization and detailed analysis of the accumulated genetic data has become a valuable asset with an increased interest in genetic screening along with the widespread recognition of precision medicine. Therefore, the aim of the study was to evaluate the mutation spectrum of the APC gene in the Korean population through analysis of the APC gene mutation tests in the most recent 15-year period. A total of 420 patients were referred for APC gene mutation tests between May 2006 and June 2020. Clinical data including clinical diagnosis, family history, colonoscopic findings, and pathologic findings were reviewed. Genomic DNA was extracted from the peripheral blood of patients suspected of having FAP and were tested with either conventional Sanger sequencing or a NGS panel test. APC gene mutations were found in 167 out of 420 patients tested (39.8%). In addition to the well-known frameshift and nonsense mutations, there were 3 cases of whole gene deletions and 3 cases of single or multiple exon deletions. Novel APC gene mutations were detected in 17 patients diagnosed with FAP. VUS was detected in 19 patients and 1 case was reclassified as a likely pathogenic variant and 6 cases were reclassified as a likely benign variant, according to the ACMG criteria. NGS panel was able to detect more APC gene mutations than the conventional direct sequencing method, in which the discrepancy was mostly associated with large deletions and somatic mosaicism which are due to limitations of the conventional sequencing method. Many of the patients with negative APC gene mutation study results who were tested before the introduction of the NGS may consider getting tested again to confirm a clinical diagnosis of FAP. Likely pathogenic mutations in BMPR1A, MUTYH, PMS1, and POLE genes were detected in 8 patients without an APC gene mutation with NGS testing. VUS was detected in a heterogeneous group of genes in 35 patients without APC gene mutations and suspicious of FAP. A frameshift variant of MSH6 gene (c.4068_4071dupGATT, p.Lys1358AspfsTer2) was detected in 7 patients with few to multiple adenomas, but showed conflicting evidence for classification. A VUS in ALK gene detected in one patient was reclassified as likely benign. Genetic testing has become an essential component in the diagnosis and management of FAP with continuous accumulation of genetic mutations in the database. Detection of previously undetectable mutations should help in the diagnosis of FAP patients without a confirmed genetic cause with the introduction of NGS panels. In conclusion, the use of NGS panels requires a robust bioinformatics algorithm for the interpretation of genetic variants, as well as periodic review of new clinical evidence and revised recommendations for accurate ACMG classification, which will be an important process in diagnosis of hereditary cancer predisposition syndromes such as FAP.

가족성 선종성 용종증은 대장암의 약 1%를 차지하는 상염색체 우성 유전질환이다. 10대 이후부터 100개 이상의 선종성 용종이 대장에 나타나며, 다발성으로 발생하여 대장암으로 진행의 위험이 있는 질환이다. 질환과 연관된 APC 유전자는 종양억제 유전자로, 염기서열의 변이 또는 결실 등으로 단백질이 조기종료되는 현상이 흔히 나타나며, 이는 다발성 용종의 발생을 억제하는 능력을 상실하게 된다. 본 연구에서는 한국인에서 가족성 선종성 용종증 의심환자 420명을 대상으로 15년간 시행한 염기서열분석법과 차세대염기서열분석법의 결과에서 돌연변이 양상을 분석하였다. 약 39.8%의 환자에서 APC 유전자 돌연변이가 발견되었고, 기존 보고된 바와 같이 틀이동돌연변이와 무의미돌연변이가 가장 흔하게 나타났다. 기보고 없는 돌연변이가 가족성 선종성 용종증 환자 17명에서 발견되었고, APC 유전자의 불확실성변이(VUS)가 19명에서 보였다. 미국의학유전학회 (ACMG)의 지침을 재분석하여 1명은 “likely pathogenic” 돌연변이로 재분류하였고, 6명은 양성변이로 재분류하였다. 차세대염기서열검사를 시행하였을 때 기존 염기서열분석법보다 더 많은 변이를 발견하였는데, 넓은 범위의 염기결손 또는 낮은 비율의 돌연변이를 찾아낼 수 없는 것은 기존 검사법에서 보일 수 있는 한계이다. 차세대염기서열검사를 시행한 환자 8명에서 APC 유전자는 정상이었지만 BMPR1A, MUTYH, PMS1, POLE 유전자의 돌연변이를 발견하였고, 추가로 35명의 환자에서 다양한 유전자에서 불확실성변이가 나타났다. 이 중 7명의 환자에서 MSH6 유전자의 틀이동변이(c.4068_4071dupGATT, p.Lys1358AspfsTer2)가 발견되었는데, 상반된 결과의 문헌들로 인해 재분류를 할 수 없었고, ALK 유전자에서 보인 불확실성변이 1개는 양성변이로 재분류되었다. 가족성 선종성 용종증 환자의 진단에는 유전자 검사가 필수적인 요소가 되었고, 차세대염기서열검사법의 도입으로 기존 검사법의 한계로 인해 검출될 수 없었던 다양한 종류의 변이를 발견할 수 있어 진단에 도움이 될 수 있다. 앞으로도 이와 같은 유전성 종양질환의 진단에 중요한 역할을 유지하기 위해서는 방대한 양의 정보를 처리하는 차세대염기서열검사법은 분석력이 입증된 생물정보학적 접근이 필요하며, 임상 정보와 ACMG 지침의 개정사항들에 대한 주기적인 재분석이 필요하다.