dos Santos W, Pereira AS, Laureano T, de Andrade ES, Reis MT, Garcia FA, Campacci N, Melendez ME, Reis RM, Galvão HC, Palmero EI. Whole-exome sequencing identifies new pathogenic germline variants in patients with colorectal polyposis. World J Gastroenterol 2025; 31(29): 104830 [DOI: 10.3748/wjg.v31.i29.104830]
Corresponding Author of This Article
Edenir I Palmero, PhD, Molecular Oncology Research Center, Barretos Cancer Hospital, Antenor Duarte Viléla, 1331, Barretos 14784-400, Brazil. edenirip@yahoo.com.br
Research Domain of This Article
Genetics & Heredity
Article-Type of This Article
Basic Study
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Wellington dos Santos, Ariane S Pereira, Thais Laureano, Edilene S de Andrade, Felipe AO Garcia, Natalia Campacci, Matias E Melendez, Rui M Reis, Edenir I Palmero, Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos 14784-400, Brazil
Ariane S Pereira, Edenir I Palmero, Department of Genetics, Brazilian National Cancer Institute - INCA, Rio de Janeiro 20231-050, Brazil
Monise T Reis, Department of Pathology, Barretos Cancer Hospital, Barretos 14784-400, Brazil
Matias E Melendez, Cell and Gene Therapy Program, Brazilian National Cancer Institute - INCA, Rio de Janeiro 20231-050, Brazil
Rui M Reis, Life and Health Sciences Research Institute (ICVS), Medical School, University of Minho, Braga 4710-057, Portugal
Rui M Reis, ICVS/3B’s, PT Government Associate Laboratory, Braga/Guimarães 4710-057, Portugal
Henrique de CR Galvão, Department of Oncogenetics, Barretos Cancer Hospital, Barretos 14784-400, Brazil
Co-first authors: Wellington dos Santos and Ariane S Pereira.
Author contributions: dos Santos W main investigation; dos Santos W and Pereira AS contributed equally to this article, they are the co-first authors of this manuscript; dos Santos W and Pereira AS wrote original draft, performed formal analysis, and validations; dos Santos W, Pereira AS, Galvão HDCR, Palmero EI, Melendez ME, and Reis RM edited the original draft; Laureano T, de Andrade ES, and Garcia FAO developed the pipeline for quality control, variants calling and in silico analysis and performed them; Campacci N was responsible for patient selection and consent; Reis MT was responsible for cases classification; dos Santos W, Pereira AS, Campacci N, and Reis MT performed the data curation; Galvão HDCR was responsible for patient and families care; Melendez ME, Reis RM, and Galvão HDCR conceptualized the project; Palmero EI supervised, conceptualized and administrated the project; Melendez ME, Reis RM, Galvão HDCR, and Palmero EI reviewed original draft; Melendez ME, Reis RM, Palmero EI, and Galvão HDCR acquired funding; and all authors thoroughly reviewed and endorsed the final manuscript.
Supported by the National Oncology Care Support Program, No. 25000.056766/2015-64.
Institutional review board statement: This study was approved by the Medical Ethics Committee of Barretos Cancer Hospital, approval No. 56164716.9.0000.5437.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: Sequence data has been deposited at the European Genome-phenome Archive, which is hosted by the EBI and the CRG, under accession number EGAD50000000842.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Edenir I Palmero, PhD, Molecular Oncology Research Center, Barretos Cancer Hospital, Antenor Duarte Viléla, 1331, Barretos 14784-400, Brazil. edenirip@yahoo.com.br
Received: January 16, 2025 Revised: February 26, 2025 Accepted: July 1, 2025 Published online: August 7, 2025 Processing time: 201 Days and 7.1 Hours
Abstract
BACKGROUND
Adenomatous polyposis confers an increased risk of developing colorectal cancer. APC and MUTYH are the major genes investigated in patients suspected of having polyposis. In addition to APC and MUTYH genes, other genes, such as POLE, POLD1, NTHL1, MBD4, MSH3 and MLH3, have recently been associated with polyposis phenotypes, conferring heterogeneity in terms of the clinical, etiological and heritable aspects of patients with polyposis.
AIM
To investigate the underlying variant landscape in patients with suspected polyposis who lack variants in the APC and MUTYH genes using whole-exome sequencing.
METHODS
Twenty-seven participants were included in the study and subjected to germline whole-exome sequencing. In addition, their clinical-pathological, personal, and family history data were collected.
RESULTS
The mean age at diagnosis was 51 years, and most participants had attenuated forms of polyposis (88.9%), with 63.0% diagnosed with a primary tumor, mostly colorectal cancer (76.5%). Among the variants identified, 17 were classified as pathogenic or likely pathogenic (in 12 participants), including variants in genes involved in the Wnt/β-catenin signaling pathway, such as ST7 L, A1CF, and DKK4, and variants in DNA-repair genes, such as NTHL1, PNKP, and PMS2, as well as a variant found at the FRK gene identified in a patient with classic polyposis at age 19 and with a family history of polyps.
CONCLUSION
This study identified novel genes potentially associated with polyposis in patients lacking germline pathogenic variants in the APC and MUTYH genes. These findings support the use of next-generation sequencing for screening, expanding the scope of polyposis-related variants beyond these two genes.
Core Tip: Adenomatous polyposis confers an increased risk of developing colorectal cancer, with the APC and MUTYH genes being the major genes involved in these cases. Other genes have been recently associated with polyposis phenotypes, conferring a heterogeneous clinical, etiological, and heritable aspects of patients with polyposis. Here, by whole exome sequencing, we investigated genes potentially related to polyposis in patients without variants in APC and MUTYH genes. The study identified 16 novel genes potentially associated with polyposis, supporting the screening by next-generation sequencing, and expanding beyond the scope of these two genes.
Citation: dos Santos W, Pereira AS, Laureano T, de Andrade ES, Reis MT, Garcia FA, Campacci N, Melendez ME, Reis RM, Galvão HC, Palmero EI. Whole-exome sequencing identifies new pathogenic germline variants in patients with colorectal polyposis. World J Gastroenterol 2025; 31(29): 104830
Colorectal cancer (CRC) is a major health issue worldwide, ranking as third most common tumor type and second in terms of the number of deaths[1], with similar trends in Brazil[2,3]. Hereditary CRC syndromes account for approximately 20% of colorectal cases, with Lynch syndrome and familial adenomatous polyposis (FAP) being the most common colorectal predisposition syndromes[4]. Approximately 1% of CRC cases are related to FAP, a syndrome that is associated with nearly 100% CRC risk if left untreated[5]. Compared with classic FAP, attenuated forms of the disease are characterized by the presence of 10-20 to 100 adenomas along the colon and rectum, usually later age of onset, and with reduced CRC risk[5].
Classic FAP is an autosomal dominant syndrome characterized by the presence of more than 100 adenomatous polyps in the colon and rectum[6] caused by germline pathogenic/Likely pathogenic (P/LP) variants in APC[5]. The position of these variants on the APC gene can influence disease severity, ranging from dozens to thousands of adenomatous polyps and different levels of lifetime risk of CRC[6]. Mono- and biallelic germline mutations in the base excision repair gene MUTYH can also lead to adenomatous polyposis[7,8]. Whole exome sequencing (WES) has also identified rare colorectal polyposis-associated genes, such as monoallelic P/LP variants in POLE and POLD1 (polymerase proofreading-associated polyposis), or biallelic P/LP variants in the NTHL1 gene (NTHL1-associated polyposis), as well as the MSH3, MBD4 and MLH3 genes, which are also involved in DNA repair mechanisms[9,10].
Despite the increasing utilization of large-scale sequencing strategies for molecular diagnosis, a significant proportion of cases remain genetically unsolved[8,11]. Moreover, the implementation of CRC screening has increased the number of patients diagnosed with multiple adenomas, highlighting the need to identify genetic causes for individuals with polyposis who remain genetically unsolved. Furthermore, a mixture of polyps with different histological classifications sometimes blurs the clinical picture, and purely based on clinical manifestations, it is often impossible to differentiate one polyposis syndrome from another[12]. To address this issue, we conducted WES in APC- and MUTYH-wild-type patients with polyposis to identify underlying germline variants in candidate genes.
MATERIALS AND METHODS
Participant selection
Patients with suspected FAP or attenuated polyposis were identified at the Department of Oncogenetics, from Barretos Cancer Hospital[13]. Following institutional routine procedures for polyposis patients, all of them were screened for germline P/LP single nucleotide variants and copy number variations in the APC and MUTYH genes via next-generation sequencing and multiplex ligation-dependent probe amplification. Patients who were negative for P/LP variants in both genes were invited to participate in this study (Supplementary Figure 1). A total of twenty-seven patients agreed to participate and provided informed consent for the study. This study was approved by the Medical Ethics Committee of Barretos Cancer Hospital, approval No. 56164716.9.0000.5437. All samples and data collected were from retrospective cases. We also collected clinical-pathological information, the personal and family history (FH) for each participant.
DNA isolation and WES
Peripheral blood genomic DNA was isolated via the QIAmp DNA Blood Mini Kit for the QIAcub automated platform (QIAGEN, Hilde, Germany). WES was performed by SOPHiA™ genetics using a Whole Exome Solution Kit (version 1) with 203058 target regions and 40907213 bp in 19682 genes on an Illumina NovaSeq platform (Illumina, San Diego, CA, United States). The mean coverage of WES was 150 ×, with 99.6%, 99.4%, 99.3% and 99.3% of sites having coverage above 10 ×, 20 ×, 20-30 × and 40-50 ×, respectively.
Reads and variant calling were performed as previously described[14]. Briefly, the read quality was assessed via FASTQC and trimmed via Cutadapt. Alignment and mapping of reads against the human genome reference (build GRCh37/hg19) was performed via the Burrows-Wheeler Aligner (BWA, version 0.7.17)[15]. Duplicated reads were removed via the Picard tool, and quality score recalibration was performed via the Genome Analysis Toolkit[16]. Genome Analysis Toolkit Haplotype Caller was used for variant calling, and variant annotation was performed via ANNOVAR[17].
We analyzed a set of 2389 genes (Supplementary Table 1), comprising cancer-related genes from the COSMIC[18], UniProt[19], and DISEASES[20] databases; hereditary cancer-related genes from commercial gene panels; genes reported in the literature [PubMed (https://pubmed.ncbi.nlm.nih.gov) and Genetics Home Reference database: (https://medlineplus.gov/genetics/)]; and genes involved in DNA repair pathways, as described by Garcia et al[14]. Variants with fewer than 30 reads, less than 25% of the variant allele fraction, and more than 1% of the minor allele frequency (based on gnomAD[21] and ABraOM[22] populational databases) were filtered out from the analysis. Manual prioritization was performed in variants classified as P/LP by ClinVar[23] or InterVar[24], loss-of-function variants (indels and nonsense), and variants of uncertain significance (VUS), by two independent researchers, following American College of Medical Genetics and Genomics (ACMG) criteria[25]. Integrative Genomics View[26] was utilized for visual inspection of classified variants. Variants classified as P/LP for a subset of hereditary cancer-related genes were confirmed by bidirectional Sanger sequencing.
Correlation between the presence of P/LP variants and clinical-pathological data
The presence of P/LP variants was correlated with the clinical and pathological data of the study participants. For categorical variables, such as gender, FH, type of polyposis, primary diagnosed tumor site, and primary diagnosed tumor stage (tumor-node-metastasis staging), Fisher’s exact test was used. To evaluate differences between the presence of P/LP variants and numerical data (age at diagnosis), we performed a non-parametric Wilcoxon test. Linear regression was also performed to calculate the odds ratio for the presence/absence of P/LP variants, with adjustments made for sex, age at polyposis diagnosis, tumor-node-metastasis tumor staging, FH, polyposis type, and primary tumor site.
Pathways enrichment analysis
To validate some findings about the P/LP variants identified and their potential importance in cellular pathways related to carcinogenesis, the Gene Ontology (GO) Enrichment Analysis was performed, available on the website https://geneontology.org/. For this, the list of genes presenting variants classified as P/LP was used, and the 'biological process’ option was chosen.
RESULTS
Characterization and clinical-pathological assessment of the participants in the study
A total of 27 patients with classic or attenuated polyposis were included in the present study. The data summary of the participants is shown in Table 1. The mean age at polyposis diagnosis was 51 years (19-69), most participants were male (51.9%), 92.6% had attenuated polyposis, and 7.4% presented multiple polyps (Table 1). Most of the participants were diagnosed with primary tumors (63.0%), mainly CRC (76.4%), and 23.5% had a second primary tumor (CRC, endometrium, breast or thyroid, Table 1). Two participants with CRC had synchronous colon tumors.
Table 1 Characterization and clinical-pathological assessment of participants of study.
A total of 394 germline variants were retained for ACMG classification after filtering for sequencing quality, allele fraction, and population frequency. The ACMG prioritization of variants resulted in 60.1% of variants classified as VUS (237 variants) and 4.3% of variants classified as P/LP (17 variants). P/LP variants were present in 12 participants, with four showing more than one variant classified as P/LP (Table 2). All VUS identified are listed in the Supplementary Table 2.
Table 2 Pathogenic and likely pathogenic variants identified in the participants of study.
Among the sixteen participants diagnosed with cancer, thirteen were diagnosed with CRC. Most of these individuals were diagnosed with both polyposis and CRC simultaneously. Additionally, four participants had other primary tumor types, specifically endometrial, breast, prostate, and thyroid cancers. Two participants had multiple primary tumors: ID 157 had CRC and breast cancer (BC), whereas ID 1963 had thyroid cancer and BC. All participants reported a FH of cancer, with approximately 63% having relatives with polyps or polyposis. Moreover, 37% of the participants had family members with CRC, and the same percentage had family members with gastric and/or intestinal cancer. Other frequently reported cancers in family histories included BC (29.6%) and prostate cancer (25.9%) (Supplementary Table 3). The detailed FH of each participant is shown in Supplementary Table 3.
P/LP variants: Association with personal and family medical histories
A total of twelve participants were identified with P/LP variants. Among them, four participants presented multiple P/LP variants (Table 2 and Figure 1). In the group of participants with more than one variant classified as P/LP, all were diagnosed with attenuated polyposis, and two were also diagnosed with CRC. The first participant (ID 1488) was diagnosed with endometrial polyps at age 50, in addition to attenuated polyposis and CRC, both of which were diagnosed at age 40. This participant reported a FH of multiple tumors. Participant ID 1488 carried one LP splicing variant in the PIDD1 gene, a LP frameshift variant in the DKK4 gene, and a heterozygous pathogenic nonsense variant in the NTHL1 gene (Table 2). Among the identified variants of VUS, a splicing variant in the GRB10 gene and a missense variant with a high pathogenic score prediction (HPSP) in the ID3 gene are noteworthy.
Figure 1 Representative plot of variants identified in patients with polyposis.
All genes harboring pathogenic (V) or likely pathogenic (IV) variants are shown (genes at the top). Genes harboring only variants of uncertain significance are shown if they have an indel, nonsense, or missense variant with a high pathogenic score (high variants of uncertain significance). ACMG: American College of Medical Genetics and Genomics.
The second participant (ID 278), also diagnosed with attenuated polyposis and CRC at age 55, reported a FH of gastrointestinal tumors and polyps. This participant carried two heterozygous LP variants in the MUC16 and TINF2 genes (Table 2), as well as a frameshift variant in the OBSCN gene classified as a VUS. Additionally, among the missense VUSs, two variants with HPSPs in the BMPR1A and CNTNAP2 genes were present.
The third participant with more than one variant classified as P/LP, participant ID 1536, was diagnosed with BC at age 58 and attenuated polyposis at age 63. She reported a FH of polyps, as well as CRC, gastric, and kidney tumors. This participant had two LP variants identified in the CD44 and TG genes (Table 2), along with five VUSs: Two frameshift variants in the BLK and TINF2 genes, a nonsense variant in the GCNT3 gene, and two missense variants with HPSP in the HBA1 and CSMD3 genes.
Finally, participant ID 1831 was diagnosed with attenuated polyposis at age 36 and reported a FH of polyps. This participant presented a LP frameshift variant in the MUC16 gene, and a splicing variant classified as pathogenic in the HLTF gene (Table 2). Additionally, this participant carried several VUSs, including the same BLK frameshift variant also identified in participant ID 1536, as well as a missense variant with a HPSP in the NSD3 gene.
Among the group of participants carrying only one variant classified as P/LP, four were diagnosed with attenuated polyposis and CRC (ID 547, ID 1165, ID 2325, and ID 2448). Participant ID 547, diagnosed at age 51, reported a FH of CRC and polyps. This participant carried a LP nonsense variant in the A1CF gene (Table 2). Additionally, we identified two VUS missense variants, one in the RAD21 gene and another with a HPSP in the MSH5 gene. Participant ID 1165, who was diagnosed with CRC at age 53, reported a FH of gastrointestinal tumors. This participant has a heterozygous splicing LP variant in the PCSK7 gene (Table 2) and carries an IGF1R missense variant classified as VUS, despite having a HPSP. Participant ID 2325 was diagnosed with CRC at age 59 and presented FH of multiple tumors, including CRC. This participant carries a LP variant in the OBSCN gene (Table 2) and has several VUS, including a missense variant with a HPSP in the NTRK1 gene. Finally, participant ID 2448, who was diagnosed with CRC at age 19, carried a pathogenic nonsense variant in the PMS2 gene (Table 2). Notably, the tumor from this patient did not show microsatellite instability.
Among the other participants who carried only one P/LP variant, none had a cancer diagnosis, although three presented loss-of-function variants. Participant ID 1490, who was diagnosed with attenuated polyposis at age 52, reported a FH of polyps. This participant carried a LP frameshift variant in the ST7 L gene (Table 2) and had two VUSs with HSPS in the SDHA and CDKN2A genes. Another participant, ID 517, who was diagnosed at age 56, carried a heterozygous LP variant in the PNKP gene (Table 2) and reported a FH of gastric, breast, and prostate tumors.
Notably, participant ID 454, who was diagnosed with classic polyposis at age 19 and has a FH of polyps, presented a LP nonsense variant in the FRK gene (Table 2), along with several VUSs, including a missense variant with a HPSP in the ESR2 gene. Finally, participant ID 907, who was diagnosed with attenuated polyposis at age 61, reported a FH of polyposis, leukemia, and lung tumors. This participant carried a heterozygous LP variant in the AHRR gene (Table 2) and two nonsense variants classified as VUS in the ABTB1 and KISS1 genes.
Correlation between presence of P/LP variants and clinical-pathological data
The presence of pathogenic/LP variants was correlated with clinical-pathological data. No correlation was observed between the presence of P/LP variants and gender (P = 0.704), FH (P > 0.999), polyposis type (P > 0.999), primary diagnosed tumor site (P = 0.876), or primary diagnosed tumor stage (P = 0.284) (Supplementary Table 4). Besides, no statistical significance was observed for the presence of P/LP variants, after adjusting for age at polyposis diagnosis and sex, with respect to FH, polyposis type, primary diagnosed tumor site, and primary diagnosed tumor stage (Supplementary Table 5). Although no significant differences were found, participants with P/LP variants were slightly younger at the time of polyposis diagnosis (mean age of 47 years vs 55 years, P = 0.16) compared to participants without P/LP variants.
Pathways enrichment analysis
GO enrichment analysis was performed on our set of genes with pathogenic variants from FAP cases, and only one significantly altered pathway was identified: Negative regulation of protein adenosine diphosphate (ADP)-ribosylation. This pathway showed a P-value of 0.0308, suggesting a potential role in the molecular mechanisms underlying FAP. These findings highlight a specific biological process that may contribute to the pathogenesis of FAP in our cohort.
DISCUSSION
APC and MUTYH are the main genes associated with adenomatous colonic polyposis[11]. While considerable knowledge exists, a significant portion of the genetic predisposition to adenomatous polyposis remains unexplained[27]. The use of high-throughput technologies, such as WES and whole genome sequencing, has enabled researchers to identify novel hereditary CRC genes, such as POLE, POLD1 and NTHL1[9,10]. In this study, to identify new candidate genes for germline polyposis predisposition, we performed WES in patients with FAP without APC or MUTYH germline PVs. In this case series, we identified 44.4% of participants who carried at least one P/LP variant in 16 DNA repair, hereditary, or cancer-related genes. Although many patients with attenuated polyposis remain without a constitutional genetic alteration that could be associated with the phenotype[28], this missing heritability may be multifactorial—resulting from the combination of moderate- or low-risk genetic variants, in conjunction with environmental or lifestyle risk factors.
Contrary to what is expected for carriers of P/LP in genes already well-associated with some tumor types[29-31], no correlation was found between the presence of a P/LP variant and FH of cancer. Furthermore, we observed no correlation or significant increase in the risk for the development of CRC or a specific type of polyposis, nor differences in the tumor stage. We also did not observe a younger age at diagnosis of polyposis in P/LP carriers, although a trend was noted for this variable. These factors may be explained by the sample size evaluated in the study, but they may also suggest that genes not yet associated with hereditary syndromes, and consequently with cancer development, may require a different screening approach than is currently recommended, based on personal and/or FH[32,33].
Pathogenic variants in the base excision repair gene NTHL1, including the p.(Gln82Ter) variant identified, have been observed in families with unexplained polyposis in homozygosity or compound heterozygosity states[34,35]. These families, characterized predominantly by adenomatous polyps, early-onset CRC, or familial CRC, exhibit a multiple primary tumor phenotype, including a high incidence of BC[34]. Similarly, our participant had her first diagnosis at age 40 (CRC and polyposis) and reported a sister with BC. Additionally, the same variant was identified in a patient with attenuated adenomatous polyposis and endometrial hyperplasia[36], which also resembled the participants reported in this study. Although biallelic germline variants in NTHL1 are associated with polyposis, carriers of monoallelic variants do not present a greater risk of polyposis or CRC[37,38]. However, mutational signatures related to the loss of NTHL1 have been observed in tumors of NTHL1 monoallelic PV carriers[38].
The same participant carrying the NTHL1 variant in this study also had two other LP variants identified in the DKK4 and PIDD1 genes. The combined presence of these variants could increase the risk of developing tumors, particularly CRC. The PIDD1 is a death domain protein, described as an effector of p53-dependent apoptosis, its inhibition has been shown to attenuate p53-mediated apoptosis, and the protein also act in the cell cycle control upon damage[39]. In this context, disruptive variants in the death domain of the protein have been shown to impair PIDD1’s ability to activate apoptosis[40], which means that damaged cells that should undergo apoptosis may survive, potentially leading to tumor development. The DKK4 gene functions as a negative regulator of the Wnt/β-catenin signaling pathway[41]. DKK4 secreted from CRC cells inactivates β-catenin in fibroblasts, resulting in restricted expansion in tumor masses and enhanced CRC metastasis in mouse models[42]. In addition, it enhances resistance to chemotherapy in CRC cells[43].
Dysregulation of the Wnt/β-catenin pathway plays a significant role in various tumor types, including polyp development and CRC[44,45]. In addition to the DKK4 gene variant, we also identified other variants in genes associated with these signaling pathways, such as A1CF. Loss of A1CF expression has been implicated in the regulation of the Wnt/β-catenin pathway[46] and is related to decreased cell proliferation and migration in gliomas[47] and to increased epithelial mesenchymal transition in normal kidney cells[48]. This gene has also been demonstrated to play a role in the tumor progression of endometrial cancer, mediating the p53/p21 signaling pathway[49]. In this study, a participant carrying the A1CF LP variant reported cases of CRC and BC in her family. A relationship between alterations in A1CF expression and tumor development and progression in BC models has already been observed[50,51].
The findings of P/LP variants in genes involved in the Wnt/β-catenin pathway are consistent with pathway GO enrichment analysis, which demonstrated significant alterations in the “negative regulation of protein ADP-ribosylation”. This is because ADP-ribosylation is involved in the activation of the Wnt signaling pathway[52]. Since it plays a role in regulating the Wnt/β-catenin pathway, its negative regulation may influence cellular processes, potentially contributing to tumor development or progression[53].
We also identified PV in PMS2, a component of the mismatch repair (MMR) pathway, associated with Lynch syndrome[54], Constitutional MMR deficiency syndrome[55], and BC[56]. Patients carrying variants in MMR genes with multiple adenomas have been reported, including variants in PMS2[57,58]. Although Lynch Syndrome is not known as a polyposis syndrome, its presentation may encompass the presence of multiple colonic polyps, resulting in an oligopolyposis phenotype, sometimes related to a type of polymerase proofreading-associated polyposis[59]. This condition is characterized by proficient MMR and stable microsatellites[59], as observed in our participant who did not exhibit microsatellite instability. Importantly, the participant in this study who was diagnosed with early-onset CRC (EO-CRC) at age 19 reported the great-grandmother with a gastrointestinal tumor, suggesting a hereditary component to the genetic cause. In this context, a deleterious heterozygous variant in PMS2 has been associated with the presence of polyps and Lynch Syndrome-associated CRC at a young age[60].
A specific case that caught our attention was that of a participant who did not report a personal or FH of cancer; however, they had a brother with polyps at age 25 and was diagnosed with classic polyposis early, at 19. This patient presented a nonsense variant identified in the FRK gene. This gene encodes the Rak tyrosine kinase, which is responsible for phosphorylating and regulating the PTEN gene, and acts as a tumor suppressor[61]. This gene is associated with high risk for BC, endometrial and thyroid tumors, and colon polyposis[62]. Lack of expression of this protein could result in no phosphorylation of PTEN, impacting its functions and leading to pathway deficiency. In addition, FRK is a tumor suppressor in glioma carcinogenesis[63], suppresses the epithelial-to-mesenchymal transition in BC[64], and its inactivation by a PV could result in tumorigenesis through different mechanisms, increasing the risk of developing cancer.
There is one group of genes that is not typically associated with cancer predisposition presented P/LP variants in our cohort. One participant with a variant in the PCSK7 gene was diagnosed with polyposis and CRC and reported a FH that included three additional CRC cases, two gastric cancer cases, and one uterine cancer case. PCSK7 is demonstrating its clinical importance in cancer biology and starting to emerge as a novel target for cancer/metastasis[65]. Additionally, it was suggested that inhibiting the protein family encoded by this gene could help prevent liver metastasis in CRC[66]. The PCSK7 gene also is downregulated in non-small cell lung cancer and may serve as a potential diagnostic biomarker, as it can distinguish early-stage non-small cell lung cancer patients from healthy individuals[67]. For patients in whom we cannot directly associate the presence of a P/LP variant with tumor development but who have strong FH, we should consider the potential contribution of other variants classified as VUS or those associated with low risk.
Another gene not typically associated with hereditary cancer, which presented an LP variant in this study, is OBSCN. The participant carrying this variant was diagnosed with polyposis and CRC. This gene encodes the protein obscurins, and both loss of mRNA expression and loss of function have been identified in cell lines from breast, skin, and CRCs[68]. Germline variants in the OBSCN gene have been reported in patients with glioblastoma multiforme[69]. Although this gene is not commonly associated with a hereditary predisposition to cancer, the presence of somatic variants in OBSCN is directly associated with prognosis[70] metastasis[71] and is considered an early driver of carcinogenesis in CRC[72].
The gene TINF2 is involved in telomere length maintenance, whose shortening has been observed in cases of EO-CRC[73]. Variants in other genes that act in telomere length maintenance and sheltering complexes (such as TINF2) are associated with susceptibility to developing EO-CRC[73]. The carrier of the variant in TINF2 (ID 278) was diagnosed with polyposis and CRC at age 55 and reported several cases of gastrointestinal cancer or polyposis in the family, some of which occurred at an early age. Heterozygous germline variants resulting in truncated forms of the TINF2 protein were found in patients with excessive telomere elongation and multiple tumor types, including CRC, BC, thyroid, and melanoma[74]. This participant also carried a variant in the MUC16 gene, as did participant ID 1831. This gene contributes to the development and progression of CRC[75]. Participant ID 1831 was not diagnosed with cancer; however, they were diagnosed with polyposis at 36 years old, and the presence of the MUC16 variant could represent a risk factor for developing cancer, especially CRC.
The discoveries using WES have been integrated into clinical practice in recent years, contributing to new discoveries related to CRC and polyposis predisposition[9,76,77]. The use of WES technology has several limitations, such as restricting variant identification in noncoding and exon flanking regions, missing potential variants in introns, regulatory regions, and rearrangements. Although we were able to identify potentially PVs in 12 of 27 individuals in the present study, the lack of somatic analysis of tumors and/or polyp lesions prevented us from investigating loss of heterozygosity. We also lacked expression data for allele variants, including those potentially affecting splice regions, which could provide additional information for pathogenic classification. Although we were unable to prove the association between the P/LP variants identified and the increased risk of polyposis, we provide insights that could greatly contribute to the identification of patients at high risk for cancer. Similarly, we provide potentially new therapeutic targets in the future (mainly for those involved in or associated with Wnt signaling[78]), as long as the associations identified in this study could be corroborated by other cohorts involving polyposis and by other parameters for variant classification, such as robust functional assays and segregation analysis.
CONCLUSION
In conclusion, we suggest 16 candidate genes potentially involved in the germline predisposition to polyposis (A1CF, AHRR, CD44, DKK4, FRK, HLTF, MUC16, NTHL1, OBSCN, PCSK7, PIDD1, PMS2, PNKP, ST7 L, TINF2, TG), which, if included in screening, could benefit several patients at risk for polyposis, CRC and other tumor types through appropriate management. To the best of our knowledge, this is the first study to analyze germline variants through WES in Brazilian cases with an unsolved molecular diagnosis of polyposis, thereby identifying new candidate genes to assess polyposis risk.
ACKNOWLEDGEMENTS
We wish to thank the Molecular Diagnostic Laboratory of Barretos Cancer Hospital for the routine molecular processing of our participant’s samples. The Researcher Support Center of Barretos Cancer Hospital for apply the informed consent and collect the clinical, sociodemographic, and histopathological data from medical records. Finally, we thank all the families and clinicians who contributed to the study.
Footnotes
Provenance and peer review: Unsolicited article; Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: Brazil
Peer-review report’s classification
Scientific Quality: Grade A, Grade A, Grade C
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Creativity or Innovation: Grade B, Grade B, Grade B
Scientific Significance: Grade B, Grade B, Grade B
P-Reviewer: Chen Y; Wu HM S-Editor: Bai Y L-Editor: A P-Editor: Wang WB
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