Technological Advances in Molecular Diagnostic Methods for Hereditary Diseases in Preconception and Prenatal Settings

Molecular Prenatal Diagnosis of Monogenic Diseases, Towards Non-invasive Procedures.

In this narrative review, we outline the historical progression of, and modern advancements in, prenatal diagnosis for monogenic diseases. Initially dependent on invasive sampling procedures, prenatal diagnosis has evolved through the integration of high-throughput molecular techniques, such as next-generation sequencing. These techniques now allow for the comprehensive detection of pathogenic variants across the genome. A major breakthrough occurred with the discovery of cell-free fetal DNA (cfDNA) in maternal plasma, originating from the placenta. This discovery enabled the development of non-invasive prenatal testing (NIPT), which is now widely used. However, non-invasive prenatal diagnosis (NIPD) for monogenic diseases took longer to develop, mainly due to the need for highly sensitive sequencing methods and sophisticated statistical analysis to determine maternal transmission. Currently, only a few countries have implemented NIPD in clinical settings. The multitude of available technologies and protocols can complicate the information provided during antenatal consultations. However, mastery of the knowledge and ethical issues surrounding NIPD will ensure optimal service and better care for pregnancies at risk of monogenic disease transmission.

Diagnostic evaluation and clinical utility of C-MoKa for prenatal genetic testing.

BACKGROUND C-MoKa (Chromosome Conformation-based Karyotyping) is a novel 3D genome mapping platform, that enables simultaneous detection of structural variations (SVs), aneuploidies, copy number variations (CNVs), and uniparental disomy (UPD) in a single test. However, its performance in routine prenatal diagnosis is still unexplored. This study aimed to comprehensively evaluate the diagnostic performance and clinical utility of C-MoKa in prenatal diagnosis, and to assess its technical concordance with standard of care (SOC) testings in prenatal diagnosis. METHODS A two-phase study was designed. In phase 1, 56 retrospective participants with known chromosomal abnormalities (CAs) were recruited, and C-MoKa was performed on their cultured amniotic fluid (AF) samples. Concordance between C-MoKa and known CAs was analyzed. In phase 2, a prospective cohort of 208 participants for prenatal diagnosis were recruited. Samples underwent C-MoKa and karyotyping (KT), with parallel chromosomal microarray analysis (CMA) or improved whole-exome sequencing (iWES). In our study, supplementary copy number probes were enhanced in iWES detection, which could provide ~ 100 kb resolution across the genome, thus either CMA or iWES platform employed was used to assess the CNV detection. Diagnostic yields and concordance were evaluated, and the discordance of SVs and CNVs were further validated by fluorescence in situ hybridization (FISH) and CNV-seq, respectively. RESULTS In the retrospective cohort with known CAs, C-MoKa achieved a 94.6% (53/56) diagnostic yield and 92.8% (52/56) concordance with KT + CNV. In the prospective cohort, its diagnostic yield was 21.2% (44/208), higher than KT (12.5%, 26/208) and CNV (18.3%, 38/208). The diagnostic yield (22.6%, 47/208) was achieved when CNV platform and C-MoKa were used together. C-MoKa exhibited concordance rates of 89.4% (186/208) with KT, 89.9% (187/208) with CNV, and 90.4% (188/208) with KT + CNV. Two SVs identified by C-MoKa but missed by KT were successfully determined by FISH, and five samples with additional CNVs identified by C-MoKa were consistently detected by CNV-seq. CONCLUSIONS Our findings demonstrate that C-MoKa is a highly effective and reliable method for prenatal diagnosis, exhibiting high concordance with SOC techniques. Compared to the conventional application of "KT + CNV" in prenatal setting, the combined use of CNV and C-MoKa appears to maximize the diagnostic yield.

Genetic Approach in Diagnosis and Follow-Up of Patients with Thalassemia: A Comprehensive Narrative Review

A tiered, genotype-informed approach—combining HPLC/CE phenotyping, targeted molecular diagnostics, genetic modifier profiling, and periodic re-evaluation—optimizes diagnostic precision and guides individualized management across the thalassemia spectrum.

Early Prenatal Detection of Fetal Genetic Mutations and Epigenetic Alterations: A Critical Appraisal of Liquid Biopsy Methodologies and Paediatric Implications

Circulating fetoplacental nucleic acids have transformed prenatal medicine within a single generation, and screening based on cell-free DNA (cfDNA) is now offered routinely in many health systems. The field is moving quickly from the detection of whole-chromosome aneuploidy towards earlier sampling, sub-chromosomal resolution, monogenic diagnosis and the interrogation of epigenetic marks, yet the evidence supporting these extensions is uneven and the downstream consequences for children are rarely examined. This critical narrative review evaluates the state of knowledge on early prenatal detection of fetal genetic variants and epigenetic alterations through maternal blood sampling, and appraises the paediatric implications of an expanding prenatal detection frontier. Literature was identified through structured searching of Europe PMC and MEDLINE, Crossref Metadata Search, OpenAlex, Semantic Scholar and targeted retrieval of professional society statements, supplemented by backward and forward citation tracking. Evidence was appraised for design adequacy, confirmatory testing, spectrum of enrolled participants, and separation of analytical from clinical validity. Three findings dominate the synthesis. First, diagnostic confidence declines sharply and predictably as the target moves from common autosomal trisomies to rare autosomal trisomies, copy number variants and single-gene conditions, and this gradient is driven more by target prevalence and by placental biology than by sequencing chemistry. Second, DNA methylation currently functions far more securely as an analytical instrument, supporting fractional quantification and tissue-of-origin deconvolution, than as a validated diagnostic target for fetal disease, and the developmental literature that motivates epigenetic prediction rests overwhelmingly on postnatal tissues rather than on prenatal plasma. Third, paediatric evidence is the weakest link in the chain: prenatal detection demonstrably alters the ascertainment and the age distribution of childhood diagnoses, but longitudinal outcome data for prenatally ascertained children remain scarce. Priorities include phenotype-linked birth cohorts of prenatally screened pregnancies, prospective validation of methylation-based classifiers against paediatric endpoints, and evaluation frameworks that treat placental discordance as clinical information rather than analytical noise.

Prenatal Whole-exome Sequencing in a Russian Clinical Cohort: Diagnostic Yield, Indications, and Spectrum of Molecular Findings

Future Trends in Cancer Prevention and Interception in Cancer Predisposition Syndromes: Leveraging Insights From High-Risk Populations.

Cancer predisposition syndromes (CPS), arising from germline pathogenic variants in cancer predisposition genes (CPGs), are increasingly recognized as major contributors to pediatric and adult malignancies. Recent genomic advances have significantly enhanced the diagnosis, surveillance, and management of CPS, providing a unique framework for cancer prevention and interception of pre-malignant lesions. Increased utilization of comprehensive germline and tumor-normal sequencing has improved diagnostic precision, allowing early identification of at-risk individuals, including those with and without a family history of cancer. Integration of genome and long-read sequencing technologies further increases diagnostic yield by detecting structural and noncoding variants. Early diagnosis strategies, such as newborn screening, have demonstrated clinical utility and cost-effectiveness in population studies, such as the TP53 R337H variant newborn screening in Brazil. Evidence-based surveillance protocols, supported by outcome data, have led to earlier tumor detection and improved survival. Novel 'liquid biopsy' approaches offer minimally invasive tools for surveillance, with the potential to detect malignancies prior to radiologic findings. Beyond early detection, emerging strategies aim to intercept tumor development. Medical prevention strategies such as metformin in Li-Fraumeni syndrome and aspirin in Lynch syndrome show promise in modifying cancer risk. Immunoprevention, particularly neoantigen-targeted vaccines in mismatch repair-deficient tumors, is an evolving frontier with preliminary evidence of immunogenicity and safety. Together, these advances represent a paradigm shift in CPS management, offering a model for precision cancer prevention. Continued international collaboration, longitudinal studies, and health economic evaluations will be critical to translating these innovations into clinical and public health practice.