Discussion
Lynch Syndrome screening aims to enhance future cancer surveillance for the patient and offer germline variant testing for their at-risk relatives. However, traditional LS screening protocols, such as those based on clinical criteria like Amsterdam I and II or Bethesda guidelines, exhibit limited sensitivity, failing to detect over 40% of LS carriers (15). The advent of NGS has revolutionized clinical laboratory practices, enabling simultaneous sequencing of multiple cancer susceptibility genes with improved efficiency (11). In our investigation, we utilized NGS to analyze individuals with a familial cancer history, successfully identifying various variants, including a pathogenic mutation in the MutL Homolog 1 (MLH1) gene, present in two young siblings.
The MLH1 gene encodes a protein essential for the DNA mismatch repair system forming heterodimer with the mismatch repair endonuclease PMS2, to generate MutL alpha complex. The encoded protein also participates in DNA damage signaling and can heterodimerize with MLH3 protein to form MutL gamma, involved in meiosis (16, 17). Disruption of the MLH1-PMS2 heterodimer compromises MMR, leading to the accumulation of replication errors and increased microsatellite instability. Also, disruption of the heterodimer MLH1-MLH3 could contribute to tumor growth by accumulating errors during meiosis (18–20).
LS is an autosomal dominant disorder caused by germline mutations in MMR genes, primarily MLH1 and MSH2. In families with LS, over 90% of the mutations are found in these two genes (10). Mutations in the MLH1 gene impair the protein’s ability to recognize and repair DNA damage. This leads to the accumulation of genetic errors and hinders apoptosis signaling, which could promote the development of cancer (16, 17). LS is associated with an increased predisposition to different cancer types, primarily colon cancer (occurring in 46–61% of individuals with a pathogenic MLH1 variant) or endometrial cancer (occurring in 34–54% of individuals with a pathogenic MLH1 variant) (2, 21). Colorectal cancer, associated with Lynch syndrome, tends to manifest at an earlier age, with an average age of diagnosis around 40 years (22).
In both subjects, the MLH1 gene variant c.350C > T (p.Thr117Met) was identified, and notably, in Individual A, this variant was present in both blood and tissue samples. According to the InSiGHT classification, the MLH1 variant is classified as class 5 (pathogenic) (23, 24). The c.350C > T variant affects a highly conserved domain in exon 4 of the MLH1 gene. This genetic change arises from cytosine to thymine substitution at position 350 in the nucleotide sequence (25, 26), causing codon 117 undergoes a pathogenic transformation, replacing a polar threonine with a hydrophobic methionine in a highly conserved domain of the MLH1 gene.
Different tests are used for the detection of LS associated with CRC, such as colonoscopy, genetic testing, MSI, immunohistochemistry (IHC) and others. Immunohistochemistry, used for screening patients with suspected LS, can detect approximately 74% of MLH1 germline mutations when using MLH1-specific antibodies (27). This test uses antibodies against four MMR proteins (MLH1, MS2, MSH6, and PMS2) to assess their expression in colorectal cancer tissue (28). For example, the biological interpretation of the MLH1 gene mutation could be shown by IHC that has demonstrated a reduction in DNA repair activity in cells carrying the Thr117Met variant which is associated with increased MSI. Functional analyses reveal reduced protein expression and compromised mismatch repair (MMR) activity linked to the disruption caused by the Thr117Met variant (29, 30). Moreover, this missense mutation likely impairs mismatch repair function by affecting the mutant protein’s ATP binding and hydrolysis, interfering with the formation of protein complex like hPMS2.
Considering that individual A has adenocarcinoma of the colon and carries a mutation in the MLH1 gene, MSI analysis of the tumor samples is recommended. This test is essential not only for confirming the diagnosis of LS but also for its prognosis, as MSI has been established as a key biomarker in CRCs. In the general population, the rate of MSI varies between 12 and 17% but has been reported to be as high as 27 to 35% in patients younger than 30 years (31). In the case of 26-year-old A, an evaluation of MSI would not only contribute to a better understanding of his diagnosis but is also essential to guide appropriate follow-up and treatment for both him and his brother.
Determination of MSI has important prognostic value, as tumors with high instability are often associated with a better clinical prognosis. In addition, MSI has been established as a predictive biomarker in the context of immunotherapy, suggesting that immune checkpoint inhibitors may be more effective in these cases. This is because tumors with a higher mutational burden, characteristic of elevated MSI, generate a greater number of neoantigens (32). These neoantigens can stimulate an immune response, making MMR-deficient tumors particularly responsive to immune checkpoint inhibitors, such as anti-PD-1/PD-L1 therapies (33–35). In our case, the MLH1 c.350C > T variant, associated with increased microsatellite instability, could potentially create a pro-inflammatory tumor microenvironment. Understanding the interplay between mismatch repair gene mutations and immune response could lead to personalized immunotherapeutic strategies for Lynch Syndrome patients (14, 15). In this context, performing MSI analysis is not only crucial for the diagnosis and treatment of individual A, but also for the preventive management and follow-up of individual B, given the possible heritable involvement of mutations in the MLH1 gene (36).
This study marks the first report of this variant in Ecuadorian patients. Nevertheless, the variant has also been previously identified in unrelated families from Uruguay and Argentina, suggesting that the c.350C > T variant may be present within the broader Latin American gene pool (37). Notably, the reported frequency of the alternative allele T is 0,000 in the Latin American population (38).
Furthermore, this variant was identified in other populations. For instance, Yanus et al. (39) studied Russian individuals with either colorectal or endometrial cancer, who were referred for LS testing, revealing the MLH1 c.350C > T variant. The results are consistent with the UMD-MMR database, which includes 55 entries for the mutation in French laboratories until 2015 (40). Moreover, another study focused on analyzing the MLH1 gene in Polish and Baltic State families, revealing the mutation in one family with colon cancer (19). Another study reported the mutation in a hereditary nonpolyposis colorectal cancer patient in Slovakia (26). These studies collectively highlight the recurrent nature of the mutation across diverse geographical regions. Despite its global prevalence, the mutation has not been previously reported in Ecuador (14).
In the tumor sample from individual A, a PTEN c.968dup variant was identified. This variant results in a frameshift and a premature stop codon, probably producing absent or truncated PTEN protein. The truncation caused by c.968dup disrupts critical domains such as C2, C-tail, and PDZ, which play pivotal roles in protein localization, stability, and interaction with other proteins (41, 42).
PTEN is a tumor suppressor that regulates the PI3K/Akt signaling pathway, which is crucial for cell growth and survival. In colorectal cancer, loss of PTEN function leads to increased activity in this pathway, promoting tumorigenesis (43, 44). Other research suggests that loss of PTEN in CRC is closely associated with increased genomic instability and worse clinical outcomes, such as more advanced disease stages and the development of liver metastasis (45, 46). In this case study, the pathogenic variants in the PTEN and MLH1 genes identified in individual A could trigger an additive effect from mutations in different genes, leading to a more aggressive disease phenotype.
However, this relationship is not consistently observed across all studies, suggesting that the role of PTEN in tumor progression may be influenced by other genetic or epigenetic factors (45–48). Moreover, in an extensive study involving 1,093 cases of CRC, PTEN mutations were detected in 43 tumors. Among these mutations, 47.3% were identified as nonsense mutations, 38.2% as frameshift mutations, 9.1% as missense mutations, and 5.5% as splice site mutations. Notably, the c.968dup variant was among the frameshift mutations closely related to CRC (49). This variability highlights the molecular complexity of CRC, particularly in cases linked to LS, and underscores the importance of further investigation to fully understand the impact of PTEN on CRC development and progression.
On the other hand, individual B carries a FH c.580G > A mutation, which has been implicated in hereditary cancer predisposition (50). The variant frequency has not been reported among the Latin American population. The c.580G > A variant, also denoted as p.Ala194Thr, is situated within the coding sequence of exon 5 in the FH gene. This genetic alteration arises from the substitution of alanine for threonine at codon 194. Notably, alanine is characterized as neutral and non-polar, whereas threonine possesses a neutral and polar nature. Consequently, advanced protein modeling techniques, coupled with the scrutiny of their physical properties, have revealed that this missense variant disrupts the functionality of FH protein (51). Despite this, the variant has been categorized as a variant of uncertain significance, primarily due to findings from RNA analysis suggesting that its impact on mRNA splicing does not significantly alter splicing patterns. Furthermore, associations have been made between this variant and other types of cancer such as pheochromocytoma, paraganglioma, and renal cell cancer. However, its low population frequency (0.00007), mainly present in individuals of European descent, makes it uninformative for assessing its pathogenicity (51, 52).
Furthermore, the PTEN protein has a physical or functional interaction with MLH1, while the FH protein does not show any interaction with PTEN or MLH1 (Supplementary Figure S2). However, the coexistence of these variants in individuals could have synergistic effects, potentially increasing their cancer risk. Even though the assay employed in this investigation covered some genes implicated in mismatch repair, it would be interesting to utilize a multigene panel encompassing an extensive spectrum of genes or SNPs associated with LS.
The genetic analysis of the ancestral components of the two brothers harboring the MLH1 c.350C > T variant, along with additional variants (PTEN c.968dup for individual A and FH c.580G > A for individual B), has provided a further understanding of the possible relationship between genetic ancestry and specific mutations associated with hereditary cancer predisposition.
Moreover, the ancestral composition of the individuals revealed a mosaic of African, European, and Native American components, as previously reported for the Ecuadorian (14). However, both siblings have high levels of Native American ancestry, which is notable given a lack of data on population specific genetic risks in this group. Although ancestry data does not directly elucidate cancer predisposition in this case, the Native American heritage may offer insights into underrepresented genetic variants relevant to Lynch Syndrome and cancer. Further studies on larger cohorts are needed to explore the relationship between ancestry and cancer risk in Latin American (14).
The MLH1 c.350C > T variant, associated with Lynch syndrome, has been extensively studied and reported in different regions, such as Latin America (37) and Europe (19, 26, 39, 40). Besides, a study compared the racial differences in MLH1 mutations between the Caucasian and East Asian races and found that this variant ranks among the top ten mutations in both ethnic groups (53). The prevalence of this mutation in individuals with predominantly Native American ancestry is of particular interest and has not been previously reported, which could provide insights into population-specific genetic risk factors. However, the PTEN and FH variants have not been previously described in other populations.
Consequently, employing multiple gene sequencing may benefit patients, especially those with personal or familial histories. The identification of individuals with hereditary cancer predispositions, such as Lynch syndrome, holds significant promise for reducing cancer incidence and mortality rates. Nonetheless, accessing comprehensive mutation screening may present challenges in certain scenarios. However, once mutations are identified, screening other family members for the same mutation can be achieved using relatively straightforward methods (15).
In this study, we used NGS panels that included the MMR genes (MLH1, MSH2, MSH6, or PMS2), identifying a pathogenic MLH1 mutation in both siblings. This finding underscores the importance of genomic testing in detecting germline mutations within families. Clinically, it enables a more precise diagnosis and treatment for Individual A, who is symptomatic, while providing necessary follow-up for Individual B, who remains asymptomatic.
A limitation of this study is the budget constraint, because NGS could not be performed on all family members. Despite this constraint, the screening process remains informative and contributes significantly to continuous patient monitoring. Consequently, the adoption of massive sequencing approaches may provide valuable insights, enabling the implementation of effective cancer prevention strategies, personalized screening protocols, and tailored therapeutic interventions to mitigate cancer-related morbidity and mortality linked to Lynch Syndrome (11, 54).
In conclusion, the ancestral components, and associated variants of the two siblings provide the first data of a mainly Native American composition and mutations related to LS and cancer predisposition. Further studies on diverse populations and larger cohorts could improve the understanding of population-specific variants, their implications, and healthcare strategies.