In Silico Deciphering of the Potential Impact of Variants of Uncertain Significance in Hereditary Colorectal Cancer Syndromes.

Colorectal cancer (CRC) ranks third in terms of cancer incidence worldwide and is responsible for 8% of all deaths globally. Approximately 10% of CRC cases are caused by inherited pathogenic mutations in driver genes involved in pathways that are crucial for CRC tumorigenesis and progression. These hereditary mutations significantly increase the risk of initial benign polyps or adenomas developing into cancer. In recent years, the rapid and accurate sequencing of CRC-specific multigene panels by next-generation sequencing (NGS) technologies has enabled the identification of several recurrent pathogenic variants with established functional consequences. In parallel, rare genetic variants that are not characterized and are, therefore, called variants of uncertain significance (VUSs) have also been detected. The classification of VUSs is a challenging task because each amino acid has specific biochemical properties and uniquely contributes to the structural stability and functional activity of proteins. In this scenario, the ability to computationally predict the effect of a VUS is crucial. In particular, in silico prediction methods can provide useful insights to assess the potential impact of a VUS and support additional clinical evaluation. This approach can further benefit from recent advances in artificial intelligence-based technologies. In this review, we describe the main in silico prediction tools that can be used to evaluate the structural and functional impact of VUSs and provide examples of their application in the analysis of gene variants involved in hereditary CRC syndromes.

Multiple colorectal adenomas syndrome: The role of MUTYH mutation and the polyps' number in clinical management and colorectal cancer risk.

BACKGROUND & AIMS: Multiple colorectal adenomas (MCRAs) can result from APC (AFAP) or biallelic MUTYH (MAP) mutations, but most patients are wild type and referred to as non-APC/MUTYH polyposis (NAMP). We aim to examine the risk of colorectal cancer (CRC) and the role of endoscopy in managing patients with MCRAs, with a specific focus on clinical features and genotype. METHODS: Records of MRCAs between 2000 and 2022 were retrospectively analysed. Patients were divided according to the genotype (MAP vs. NAMP) and the number of categorised polyps' burden (group 1: 10-24, group 2: 25-49, and group 3: 50-99 adenomas). Predictors of outcome were CRC-free survival (CRC-FS) and Surgery free-survival (S-FS). RESULTS: 220 patients were enrolled (NAMP n = 178(80.0%)). CRC at diagnosis was more frequent in group 3 (p = 0.01), without significant differences between the genotypes (p = 0.20). At a follow-up of 83(41-164) months, 15(7%) patients developed CRC during surveillance. CRC-FS was not correlated to genotype (p = 0.07) or polyps' number (p = 0.33), while S-FS was similar in MAP and NAMP (p = 0.22) and lower in groups 2 and 3 (p = 0.0001). CONCLUSIONS: MAP and NAMP have the same CRC risk and no difference in treatment. Endoscopic surveillance compared favorably with surgery in avoiding CRC risk, even in patients with more severe colorectal polyposis.

PTEN hamartoma tumour syndrome: case report based on data from the Iranian hereditary colorectal cancer registry and literature review.

BACKGROUND: PTEN hamartoma tumour syndrome (PHTS) is a rare hereditary disorder caused by germline pathogenic mutations in the PTEN gene. This study presents a case of PHTS referred for genetic evaluation due to multiple polyps in the rectosigmoid area, and provides a literature review of PHTS case reports published between March 2010 and March 2022. CASE PRESENTATION: A 39-year-old Iranian female with a family history of gastric cancer in a first-degree relative presented with minimal bright red blood per rectum and resistant dyspepsia. Colonoscopy revealed the presence of over 20 polyps in the rectosigmoid area, while the rest of the colon appeared normal. Further upper endoscopy showed multiple small polyps in the stomach and duodenum, leading to a referral for genetic evaluation of hereditary colorectal polyposis. Whole-exome sequencing led to a PHTS diagnosis, even though the patient displayed no clinical or skin symptoms of the condition. Further screenings identified early-stage breast cancer and benign thyroid nodules through mammography and thyroid ultrasound. METHOD AND RESULTS OF LITERATURE REVIEW: A search of PubMed using the search terms "Hamartoma syndrome, Multiple" [Mesh] AND "case report" OR "case series" yielded 43 case reports, predominantly in women with a median age of 39 years. The literature suggests that patients with PHTS often have a family history of breast, thyroid and endometrial neoplasms along with pathogenic variants in the PTEN/MMAC1 gene. Gastrointestinal polyps are one of the most common signs reported in the literature, and the presence of acral keratosis, trichilemmomas and mucocutaneous papillomas are pathognomonic characteristics of PHTS. CONCLUSION: When a patient presents with more than 20 rectosigmoid polyps, PHTS should be considered. In such cases, it is recommended to conduct further investigations to identify other potential manifestations and the phenotype of PHTS. Women with PHTS should undergo annual mammography and magnetic resonance testing for breast cancer screening from the age of 30, in addition to annual transvaginal ultrasounds and blind suction endometrial biopsies.

Promotion Effects of Smoking in Polyp Development in Monozygotic Twins with Atypical Colorectal Polyposis.

Smoking is a known risk factor for the development of colorectal polyps. Even in familial adenomatous polyposis and serrated polyposis syndrome, smoking is a risk factor of the development of polyps. Here, we report a case of monozygotic twins with atypical colorectal polyposis showing lots of hyperplastic polyps and adenomas and describe how the polyposis developed differently in the brothers based on the presence or absence of smoking. The case was of a set of monozygotic male twins, and the twins were in their 50s. The younger brother smoked 40 cigarettes a day since he was 16 years old. The older brother had smoked about 25 cigarettes a day since he was 16 years old but stopped smoking after he was diagnosed with polyposis. As we previously reported, we managed to remove polyps as much as possible from both twins without surgery. The median number of removed polyps (IQR: 25-75%) per colonoscopy for 20 years was 9.0 (3.5-14.8) in the older brother and 20.5 (7.5-35.5) in the younger brother. There was a significant difference between the twins (p < 0.01). Additionally, genetic tests found that the twins carried a rare missense variant of BRCA2, and this variation has not been previously reported. In conclusion, these monozygotic twins with atypical colorectal polyposis showing a new variant of BRCA2 suggest that smoking is related to the development of colorectal polyps. Further analysis will be required for the identified BRCA2 variant in possible involvement in the development of atypical polyposis.

Use of sanger and next-generation sequencing to screen for mosaic and intronic APC variants in unexplained colorectal polyposis patients.

In addition to classic germline APC gene variants, APC mosaicism and deep intronic germline APC variants have also been reported to be causes of adenomatous polyposis. In this study, we investigated 80 unexplained colorectal polyposis patients without germline pathogenic variants in known polyposis predisposing genes to detect mosaic and deep intronic APC variants. All patients developed more than 50 colorectal polyps, with adenomas being predominantly observed. To detect APC mosaicism, we performed next-generation sequencing (NGS) in leukocyte DNA. Furthermore, using Sanger sequencing, the cohort was screened for the following previously reported deep intronic pathogenic germline APC variants: c.1408 + 731C > T, p.(Gly471Serfs*55), c.1408 + 735A > T, p.(Gly471Serfs*55), c.1408 + 729A > G, p.(Gly471Serfs*55) and c.532-941G > A, p.(Phe178Argfs*22). We did not detect mosaic or intronic APC variants in the screened unexplained colorectal polyposis patients. The results of this study indicate that the deep intronic APC variants investigated in this study are not a cause of colorectal polyposis in this Dutch population. In addition, NGS did not detect any further mosaic variants in our cohort.

Intensive surveillance endoscopy for multiple gastrointestinal tumors in a patient with constitutional mismatch repair deficiency: case report.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) is an extremely rare autosomal recessive hereditary disease characterized by the absence of mismatch repair gene activity from birth, which results in brain tumors, colonic polyposis, gastrointestinal cancers, and lymphomas later in life. An aggressive approach, including colectomy or proctocolectomy, is recommended for the treatment of colorectal cancer. Additionally, partial colectomy with subsequent endoscopic surveillance may be an alternative strategy due to poor patient's condition, although there is no evidence of surveillance endoscopy after partial colectomy for CMMRD. CASE PRESENTATION: A 13-year-old male patient with a history of T-lymphoblastic lymphoma underwent total gastrointestinal endoscopy, which revealed rectal cancer, colorectal polyposis, and duodenal adenoma. Differential diagnosis included constitutional mismatch repair deficiency according to its scoring system and microsatellite instability, and subsequent germline mutation testing for mismatch repair genes confirmed the diagnosis of constitutional mismatch repair deficiency based on a homozygous mutation in mutS homolog 6 (MSH6). The patient and his family refused colectomy due to the high risk of malignancies other than colorectal cancer, which could require radical surgery. Therefore, the patient underwent low anterior resection of the rectosigmoid colon for rectal cancer and intensive surveillance endoscopy for the remaining colon polyposis. During the 3-year period after initial surgery, 130 polyps were removed and the number of polyps gradually decreased during 6-months interval surveillance endoscopies, although only one polyp was diagnosed as invasive adenocarcinoma (pT1). CONCLUSIONS: Our experience of short surveillance endoscopy illustrates that this strategy might be one of options according to patient's condition.

Definition and management of colorectal polyposis not associated with APC/MUTYH germline pathogenic variants: AIFEG consensus statement.

An expert consensus panel convened by the Italian Association for Inherited and Familial Gastrointestinal Tumors (Associazione Italiana per lo Studio della Familiarità ed Ereditarietà dei Tumori Gastrointestinali, AIFEG) reviewed the literature and agreed on a number of position statements regarding the definition and management of polyposis coli without an identified pathogenic mutation on the APC or MUTYH genes, defined in the document as NAMP (non-APC/MUTYH polyposis).

Low frequency of POLD1 and POLE exonuclease domain variants in patients with multiple colorectal polyps.

BACKGROUND: Germline mutations affecting the exonuclease domains of POLE and POLD1 predispose to colorectal adenomas and carcinoma. Here, we aimed to screen the exonuclease domains to find the genetic causes of multiple colorectal polyps in unexplained cases. METHODS: Using a custom next-generation sequencing panel, we sequenced the exonuclease domains of POLE and POLD1 in 332 index patients diagnosed with multiple colorectal polyps without germline alteration in colorectal polyposis predisposing genes. RESULTS: We identified two variants of unknown significance. One germline POLD1 c.961G>A, p.(Gly321Ser) variant was found in two cases. The first patient was diagnosed with multiple polyps at age 35 and colorectal cancer (CRC) at age 37, with no known family history of CRC. The second patient was diagnosed with CRC at age 44 and cumulatively developed multiple polyps; this patient had two sisters with endometrial cancer who did not carry the variant. Furthermore, we identified a novel POLD1 c.955 T>G, p.(Cys319Gly) variant in a patient diagnosed with multiple colorectal adenomas at age 40. Co-segregation analysis showed that one sister who cumulatively developed multiple adenomas from age 34, and another sister who developed CRC at age 38 did not carry the variant. We did not identify pathogenic variants in POLE and POLD1. CONCLUSION: This study confirms the low frequency of causal variants in these genes in the predisposition for multiple colorectal polyps, and also establishes that these genes are a rare cause of the disease.

The role of inherited genetic variants in colorectal polyposis syndromes.

Colorectal carcinoma (CRC) is the third most common cancer in men and the second most common cancer in women across the world. Most CRCs occur sporadically, but in 15-35% of cases, hereditary factors are important. Some patients with an inherited predisposition to CRC will be diagnosed with a "genetic polyposis syndrome" such as familial adenomatous polyposis (FAP), MUTYH-associated polyposis (MAP), polymerase proofreading associated polyposis (PPAP), NTHL1-associated polyposis, MSH3-associated polyposis or a hamartomatous polyposis syndrome. Individuals with ≥10 colorectal polyps have traditionally been referred for genetic diagnostic testing to identify APC and MUTYH mutations which cause FAP and MAP respectively. Mutations are found in most patients with >100 adenomas but in only a minority of those with 10-100 adenomas. The reasons that diagnostic laboratories are not identifying pathogenic variants include mutations occurring outside of the open reading frames of genes, individuals exhibiting generalized mosaicism and the involvement of additional genes. It is important to identify patients with an inherited polyposis syndrome, and to define the mutations causing their polyposis, so that the individuals and their relatives can be managed appropriately.

Hereditary forms of colorectal cancer. / Formas hereditarias de cáncer colorrectal.

Colorectal cancer is one of the most frequent neoplasms in western countries; it is the third most common cancer in men after prostate and lung cancer and the second most common in women after breast cancer. Colorectal cancer is usually sporadic but in a small proportion is hereditary. The genetic cause is well established, allowing pre-symptomatic diagnosis in at-risk relatives. The present article reviews the most novel findings presented at the latest meeting of the American Gastroenterological Association on hereditary forms of colorectal cancer, especially Lynch syndrome and MUTYH-associated polyposis, as well as diverse organisational aspects that can favour the correct management of these patients and their relatives.

Colorectal Adenomatous Polyposis: Heterogeneity of Susceptibility Gene Mutations and Phenotypes in a Cohort of Italian Patients.

AIMS: Colorectal adenomatous polyposis entailing cancer predisposition is caused by constitutional mutations in different genes. APC is associated with the familial adenomatous polyposis (FAP/AFAP) and MUTYH with the MUTYH-associated polyposis (MAP), while POLE and POLD1 mutations cause the polymerase proofreading-associated polyposis (PPAP). METHODS: We screened for mutations in patients with multiple adenomas/FAP: 121 patients were analyzed for APC and MUTYH mutations, and 36 patients were also evaluated for POLE and POLD1 gene mutations. RESULTS: We found 20 FAP/AFAP, 15 MAP, and no PPAP subjects: pathogenic mutations proved to be heterogeneous, and included 5 APC and 1 MUTYH novel mutations. The mutation detection rate was significantly different between patients with 5-100 polyps and those with >100 polyps (p = 8.154 × 10-7), with APC mutations being associated with an aggressive phenotype (p = 1.279 × 10-9). Mean age at diagnosis was lower in FAP/AFAP compared to MAP (p = 3.055 × 10-4). Mutation-negative probands showed a mean age at diagnosis that was significantly higher than FAP/AFAP (p = 3.46986 × 10-7) and included 45.3% of patients with <30 polyps and 70.9% of patients with no family history. CONCLUSIONS: This study enlarges the APC and MUTYH mutational spectra, and also evaluated variants of uncertain significance, including the MUTYH p.Gln338His mutation. Moreover this study underscores the phenotypic heterogeneity and genotype-phenotype correlations in a cohort of Italian patients.

Efficacy and safety of eflornithine (CPP-1X)/sulindac combination therapy versus each as monotherapy in patients with familial adenomatous polyposis (FAP): design and rationale of a randomized, double-blind, Phase III trial.

BACKGROUND: Molecular studies suggest inhibition of colorectal mucosal polyamines (PAs) may be a promising approach to prevent colorectal cancer (CRC). Inhibition of ornithine decarboxylase (ODC) using low-dose eflornithine (DFMO, CPP-1X), combined with maximal PA export using low-dose sulindac, results in greatly reduced levels of normal mucosal PAs. In a clinical trial, this combination (compared with placebo) reduced the 3-year incidence of subsequent high-risk adenomas by >90 %. Familial Adenomatous Polyposis (FAP) is characterized by marked up-regulation of ODC in normal intestinal epithelial and adenoma tissue, and therefore PA reduction might be a potential strategy to control progression of FAP-related intestinal polyposis. CPP FAP-310, a randomized, double-blind, Phase III trial was designed to examine the safety and efficacy of sulindac and DFMO (alone or in combination) for preventing a clinically relevant FAP-related progression event in individuals with FAP. METHODS: Eligible adults with FAP will be randomized to: CPP-1X 750 mg and sulindac 150 mg, CPP-1X placebo and sulindac 150 mg, or CPP-1X 750 mg and sulindac placebo once daily for 24 months. Patients will be stratified based on time-to-event prognosis into one of the three treatment arms: best (ie, longest time to first FAP-related event [rectal/pouch polyposis]), intermediate (duodenal polyposis) and worst (pre-colectomy). Stage-specific, "delayed time to" FAP-related events are the primary endpoints. Change in polyp burden (upper and/or lower intestine) is a key secondary endpoint. DISCUSSION: The trial is ongoing. As of February 1, 2016, 214 individuals have been screened; 138 eligible subjects have been randomized to three treatment groups at 15 North American sites and 6 European sites. By disease strata, 26, 80 and 32 patients are included for assessment of polyp burden in the rectum/pouch, duodenal polyposis and pre-colectomy groups, respectively. Median age is 40 years; 59 % are men. The most common reasons for screening failure include minimal polyp burden (n = 22), withdrawal of consent (n = 9) and extensive polyposis requiring immediate surgical intervention (n = 9). Enrollment is ongoing. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov ( NCT01483144 ; November 21, 2011) and the EU Clinical Trials Register( EudraCT 2012-000427-41 ; May 15, 2014).

Colorectal adenomatous polyposis syndromes: Genetic determinism, clinical presentation and recommendations for care.

Colorectal adenomatous polyposis constitutes a diverse group of disorders with different modes of inheritance. Molecular diagnosis of this condition has become more complex. In fact, somatic mosaicism for APC mutations now appears to be more frequent than previously thought and rare germline alterations of this gene may be implicated in patients tested negative for "classical" APC mutations (point mutations and large genomic rearrangements). Moreover, the knowledge concerning several aspects of the MUTYH-associated polyposis has improved since its first description in 2002 and germline mutations in new genes have recently been implicated in some cases of unexplained adenomatous polyposis. Genetic testing in probands and their relatives should be conducted in the context of pre- and post-test genetic counseling. The recent advent of New Generation Sequencing (NGS) techniques affords the opportunity to rapidly screen patients for a comprehensive panel of colorectal cancer susceptibility genes in a cost-effective fashion. This type of approach will probably replace the classical sequential approach based on clinical presumptive diagnoses in the near future. The risk of colorectal cancer is very high in affected patients in the absence of appropriate care. Clinical management is complex and should be provided in centers with special expertise in these diseases. This review focuses on the various colorectal adenomatous polyposis syndromes with special attention to more innovative and important aspects.

Mutation analysis of MUTYH in Japanese colorectal adenomatous polyposis patients.

Germline MUTYH mutations were investigated in 14 Japanese colorectal polyposis patients without germ line adenomatous polyposis coli (APC) gene mutations. Three patients had a heterozygous IVS10-2A>G MUTYH mutation. The onset of MUTYH-associated polyposis (MAP) occurs later than that of familial adenomatous polyposis with germline APC mutation. Thus, we compared the carrier frequency of MUTYH IVS10-2A>G heterozygote in the APC mutation negative cases with that in 115 controls over 70 years of age who showed no apparent clinical manifestations of cancer and claimed that they had no history of cancer at the time of enrollment. The frequency of IVS10-2A>G heterozygote in APC germline mutation negative polyposis patients was significantly higher than control subject (p = 0.012, Chi square test). Although the sample size is still too small to conclude, the IVS10-2A>G MUTYH heterozygote might add to the risk of developing germline APC mutation negative polyposis.

Prophylactic colectomy: Rationale, indications, and approach.

Discerning the genetics of hereditary colorectal cancer syndromes has enabled a more individualized approach to preventing and treating colorectal cancers in affected patients and families. As these syndromes carry significant risk of colorectal cancer (nearly 100% in some cases), prophylactic colectomy is an important option for preventing cancer. This article addresses the rationale and indications for prophylactic colectomy in hereditary colorectal cancer syndromes, and provides insight into the details of surgical management.

Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis.

AIM: To investigate the suppressive activity of MUTYH variant proteins against mutations caused by oxidative lesion, 8-hydroxyguanine (8OHG), in human cells. METHODS: p.R154H, p.M255V, p.L360P, and p.P377L MUTYH variants, which were previously found in patients with colorectal polyposis and cancer, were selected for use in this study. Human H1299 cancer cell lines inducibly expressing wild-type (WT) MUTYH (type 2) or one of the 4 above-mentioned MUTYH variants were established using the piggyBac transposon vector system, enabling the genomic integration of the transposon sequence for MUTYH expression. MUTYH expression was examined after cumate induction using Western blotting analysis and immunofluorescence analysis. The intracellular localization of MUTYH variants tagged with FLAG was also immunofluorescently examined. Next, the mutation frequency in the supF of the shuttle plasmid pMY189 containing a single 8OHG residue at position 159 of the supF was compared between empty vector cells and cells expressing WT MUTYH or one of the 4 MUTYH variants using a supF forward mutation assay. RESULTS: The successful establishment of human cell lines inducibly expressing WT MUTYH or one of the 4 MUTYH variants was concluded based on the detection of MUTYH expression in these cell lines after treatment with cumate. All of the MUTYH variants and WT MUTYH were localized in the nucleus, and nuclear localization was also observed for FLAG-tagged MUTYH. The mutation frequency of supF was 2.2 × 10(-2) in the 8OHG-containing pMY189 plasmid and 2.5 × 10(-4) in WT pMY189 in empty vector cells, which was an 86-fold increase with the introduction of 8OHG. The mutation frequency (4.7 × 10(-3)) of supF in the 8OHG-containing pMY189 plasmid in cells overexpressing WT MUTYH was significantly lower than in the empty vector cells (P < 0.01). However, the mutation frequencies of the supF in the 8OHG-containing pMY189 plasmid in cells overexpressing the p.R154H, p.M255V, p.L360P, or p.P377L MUTYH variant were 1.84 × 10(-2), 1.55 × 10(-2), 1.91 × 10(-2), and 1.96 × 10(-2), respectively, meaning that no significant difference was observed in the mutation frequency between the empty vector cells and cells overexpressing MUTYH mutants. CONCLUSION: The suppressive activities of p.R154H, p.M255V, p.L360P, and p.P377L MUTYH variants against mutations caused by 8OHG are thought to be severely impaired in human cells.