Chromosomal aneuploidy, particularly sex chromosomal aneuploidies (SCAs), present significant challenges in their screening despite their prevalence among liveborn infants. Traditional prenatal screening methods, such as karyotyping, invasive procedures like amniocentesis, and newborn screening, have limitations in terms of time, invasiveness, and associated risks, thus arising ethical considerations. The emergence of Nextgeneration sequencing (NGS) and Third-generation sequencing (TGS) technologies has revolutionized genetic diagnostics, offering comprehensive and accurate genetic profiling capable of detecting chromosomal aberrations. Despite the current dominance of NGS, mainly Illumina, in DNA-based molecular diagnostics, TGS, namely Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT), present a promising future with their ability to perform long-read real-time sequencing. Recent advancements in ONT chemistry and basecalling algorithms have significantly reduced error rates and increased throughput, making ONT an accurate, cost-effective TGS platform. Nevertheless, the transition from conventional to advanced diagnosis methods raises ethical concerns, especially regarding the disclosure of genetic information and the ethical implications of prenatal and postnatal diagnosis, while undiagnosed subjects are not getting the right medical nor genetic support.

This thesis aimed to address these concerns by developing a postnatal, non-invasive diagnostic test for genetic disorders, with focus on SCA. For this purpose, a showcase study focusing on Klinefelter Syndrome (KS), the most prevalent, yet undiagnosed SCA, was conducted. Through targeted amplicon sequencing, our assay, Screening Chromosomal ANeuploidies (SCAN), demonstrated the potential for postnatal, low-cost mass screening utilizing ONT, presenting high accuracy, specificity, and sensitivity. Additionally, AI-software allowing for real-time data processing and analysis was developed, acting as a decision-supporting tool for the screening of genetic disorders focusing on SCAs. In 2021, by combining the aforementioned wet-lab protocol and an automated AI solution for real-time data analysis, our assay, SCAN, received IVD certification, becoming the world’s first IVD-certified genetic test utilizing nanopore sequencing.

The method was further extrapolated for the detection of the second most prevalent SCA, namely Turner Syndrome (TS), demonstrating the possibility to expand the assay and pipelines for the detection of other syndromes. Given its time- and cost- effectiveness, together with its feasible potential for the simultaneous detection of various aneuploidies, SCAN represents a possibility of changing genetic diagnostics through the integration of ONT and AI for postnatal mass screening.

Furthermore, the application of screening methods, such as SCAN, for the detection of SCAs like KS and TS is discussed, emphasizing the importance of early diagnosis for improving patient care and outcomes, including the need for comprehensive genetic counseling and multidisciplinary care. As an extension of this, the importance of the transition of children with TS to young adults in the healthcare system is stressed in the third manuscript of the present thesis. Finally, in the fourth manuscript, the potential of ONT for sequencing short tandem repeats (STRs) is explored in a literature review, offering an insight into implications and future research directions.

In conclusion, in this multidisciplinary thesis, a comprehensive understanding of the challenges associated with undiagnosed SCAs and the utilization of nanopore sequencing in combination with AI.