Rapid Whole Genome Sequencing (rWGS) represents a valuable exploration in critically ill pediatric patients. Early diagnosis allows care to be adjusted. We evaluated the feasibility, turnaround time (TAT), yield, and utility of rWGS in Belgium. Twenty-one unrelated critically ill patients were recruited from the neonatal intensive care units, the pediatric intensive care unit, and the neuropediatric unit, and offered rWGS as a first tier test. Libraries were prepared in the laboratory of human genetics of the University of Liège using Illumina DNA PCR-free protocol. Sequencing was performed on a NovaSeq 6000 in trio for 19 and in duo for two probands. The TAT was calculated from the sample reception to the validation of results. Clinical utility data were provided by treating physicians. A definite diagnosis was reached in twelve (57.5%) patients in 39.80 h on average (range: 37.05–43.7). An unsuspected diagnosis was identified in seven patients. rWGS guided care adjustments in diagnosed patients, including a gene therapy, an off-label drug trial and two condition-specific treatments. We successfully implemented the fastest rWGS platform in Europe and obtained one of the highest rWGS yields. This study establishes the path for a nationwide semi-centered rWGS network in Belgium.

Infertility in couples is a common problem, with both female and male factors contributing to similar extents. Severe, congenital disorders affecting fertility are, however, rare. While folliculogenesis and spermatogenesis are generally orchestrated via different mechanisms, some genetic anomalies can impair both female and male gametogenesis. Minichromosome maintenance complex component 9 (MCM9) is involved in DNA repair and mutations of the MCM9 gene have been previously reported in females with premature ovarian insufficiency (POI). MCM9 is also an emerging cancer risk gene. We performed next-generation and Sanger sequencing of fertility and related genes and hormonal and imaging studies in a kindred whose members had POI and disordered spermatogenesis. We identified a homozygous pathogenic MCM9 variant, c.394C>T (p.Arg132*) in three sisters affected by POI due to ovarian dysgenesis and their brother who had normal pubertal development but suffered from non-obstructive azoospermia. Testicular biopsy revealed Sertoli cell-only testicular histopathology. No evidence of early onset cancer was found in the homozygotic family members, but they were all young (<30 years) at the time of the study. In the male patient the homozygous MCM9 variant led to normal pubertal development and hormonal levels but caused a Sertoli-cell-only syndrome with non-obstructive azoospermia. In the homozygous females studied, the clinical, hormonal, and gonadal phenotypes revealed ovarian dysgenesis consistent with previous reports. Active screening for potential colorectal and other cancer risks in the homozygotic MCM9 subjects has been instigated.

Newborn screening (NBS) has evolved significantly since its inception, yet many treatable rare diseases remain unscreened due to technical limitations. The BabyDetect study used gene panel sequencing to expand NBS to treatable conditions not covered by conventional biochemical screening. We present here the analytical validation of this workflow, assessing sensitivity, precision, and reproducibility using dried blood spots from newborns. We implemented strict quality control thresholds for sequencing, coverage, and contamination, ensuring high reliability. Longitudinal monitoring confirmed consistent performance across more than 5900 samples. Automation of DNA extraction improved scalability, and a panel redesign enhanced the coverage and selection of targeted regions. By focusing on known pathogenic/likely pathogenic variants, we minimized false positives and maintained clinical actionability. Our findings demonstrate that gene panel sequencing-based NBS is feasible, accurate, and scalable, addressing critical gaps in current screening programs.

Newborn Screening (NBS) has evolved significantly since its inception, yet many treatable rare diseases remain unscreened due to technical limitations. The BabyDetect study used a targeted next-generation sequencing (tNGS) panel to expand NBS to treatable conditions not covered by conventional screening. We present here the analytical validation of this workflow, assessing sensitivity, precision, and reproducibility using dried blood spots from newborns. We implemented strict quality control thresholds for sequencing, coverage, and contamination, ensuring high reliability. Longitudinal monitoring confirmed consistent performance across more than 5,900 samples. Automation of DNA extraction improved scalability, and a panel redesign enhanced the coverage and selection of targeted regions. By focusing on known pathogenic/likely pathogenic variants, we minimized false positives and maintained clinical actionability. Our findings demonstrate that tNGS-based NBS is feasible, accurate, and scalable, addressing critical gaps in current screening programs.