Optimizing mixed sample analysis as a step to comprehensive desease screening: a pilot study

Background: Lynch Syndrome (LS) is an autosomal dominant hereditary syndrome associated with a diverse range of cancer types. Despite being one of the most prevalent hereditary cancer syndromes, the detection of LS remains challenging due to the absence of well-defined diagnostic criteria which would be able to select all patients who should undergo testing for LS and the limitations of existing screening methods. The implementation of an efficient screening program capable of accurately detecting the majority of LS cases remains a topic of continuous discussion in the scientific literature, with recent studies emphasizing the significance of a universal screening program. Methods: Our study aimed to develop and optimize a cost-effective universal screening method for detecting mutation in the mismatch repair (MMR) genes through mixed sample analysis. We tested five approaches in terms of the use of biological material and the analysis of mixed samples. Results: Each approach successfully detected a specific Lynch-associated pathogenic variant in mixed in the pooled samples with frequency 5.00%, with the lowest allelic fraction recorded at 3.04%. Approach 2, which involved isolating DNA from each patient individually, demonstrated the highest average allelic fraction (7.04%). However, considering financial and time requirements, approach 1, where DNA was isolated only after mixing aliquots of whole blood, proved to be the most favorable. Conclusion: The findings of our study present a promising opportunity to improve LS detection. The identification of LS not only has the potential to prevent cancer-related morbidity and mortality but also facilitates continued progress in understanding the primary prevention of cancer. ### Competing Interest Statement The authors Lucia Krasnicanova, Natalia Forgacova, and Vanda Repiska declare no conflicts of interest that could influence the work reported in this paper. Tatiana Sedlackova, Jaroslav Budis, Juraj Gazdarica, and Tomas Szemes are the employees of Geneton Ltd. which is involved in numerous research and development efforts dedicated to adapting new technologies to better understand genomic data and facilitate their implementation in effective and reliable patient care. ### Funding Statement This work was funded by the Operational program Integrated Infrastructure within the project: Creation of nuclear herds of dairy cattle with a requirement for high health status through the use of genomic selection, innovative biotechnological methods, and optimal management of breeding (NUKLEUS), ITMS code 313011V387, co-financed by the European Regional Development Fund. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethical Committee of the Bratislava Self-Governing Region (Sabinovska ul.16, 820 05 Bratislava), on 30 November 2020 under the decision ID 09834/2020/HF gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.