Bart de Koning

Genome‐wide non‐invasive prenatal testing (NIPT) is a powerful tool for prenatal detection of the common aneuploidies causing Down‐, Edwards‐, and Patau syndrome. Its genome‐wide reach also enables the detection of unbalanced structural chromosomal abnormalities. We report a case where NIPT indicated a ~25 Mb terminal deletion of the long arm of chromosome 16, later confirmed to be mosaic in maternal blood and buccal cells (in 35%–45% of cells). Remarkably, the same mosaic deletion was seen in the child, who was born healthy at term after an uneventful pregnancy. Further analysis using bromodeoxyuridine (BrdU)‐induced cultures revealed that the deletion originated from the expression of a rare autosomal fragile site (RFS), FRA16B, resulting in enhanced chromosomal fragility at 16q21. Rather than inheriting the maternal deletion, the child had inherited the maternal RFS, causing increased fragility at 16q21 in both mother and child. Employing long‐read sequencing (LRS), we characterized the molecular structure of this RFS. FRA16B is an expanded repeat region of > 20 kb in size, comprising over 700 AT‐rich minisatellite repeats. We resolved both distal (35‐mer) and proximal (28‐mer) repeat motifs at nucleotide precision. This uncovered striking molecular parallels between FRA16B and FRA10B, an RFS that is also associated with clinically irrelevant deletions found occasionally in NIPT. Our case underscores the importance of cautious interpretation of 16q21 terminal deletions in NIPT in order to avoid unnecessary invasive prenatal testing, as their presence reflects maternal and/or fetal fragile site instability rather than a pathogenic chromosome abnormality.

Background: Amelogenesis imperfecta (AI) is a rare genetic disorder causing tooth enamel defects. AI has been classified into 14 different clinical subtypes with different modes of inheritance. In this study, we performed whole‐exome sequencing to identify the causative gene defect in a large Dutch family with autosomal dominant hypocalcified AI (ADHCAI).Methods: Whole‐exome sequencing (WES) was performed on genomic DNA of the proband with AI. We focused on eight candidate genes known to be involved in inherited autosomal dominant AI. Sanger sequencing was used to confirm the selected exome candidate variant. Additionally, genotype and phenotype analyses were performed in the selected affected and non‐affected individuals and compared according to previously listed literature for this candidate gene of the proband.Results: The clinical phenotype of the affected individuals showed a generalized and extensive enamel defect of all teeth. In the exome dataset of the proband, a novel nonsense variant in FAM83H, c.1055C>A p.(Ser352*) was detected, which was verified by conventional Sanger sequencing. Co‐segregation analysis confirmed that the variant was present in all affected individuals and not in unaffected individuals.Conclusion: A novel pathogenic, protein‐truncating variant was detected in FAM83H, a gene with similar truncating variants known to be associated with ADHCAI.

High-throughput sequencing technologies have increasingly led to discovery of disease-causing genetic variants, primarily in postnatal multi-cell DNA samples. However, applying these technologies to preimplantation genetic testing (PGT) in nuclear or mitochondrial DNA from single or few-cells biopsied from in vitro fertilised (IVF) embryos is challenging. PGT aims to select IVF embryos without genetic abnormalities. Although genotyping-by-sequencing (GBS)-based haplotyping methods enabled PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A), they are labour intensive, only partially cover the genome and are troublesome for difficult loci and consanguineous couples. Here, we devise a simple, scalable and universal whole genome sequencing haplarithmisis-based approach enabling all forms of PGT in a single assay. In a comparison to state-of-the-art GBS-based PGT for nuclear DNA, shallow sequencing-based PGT, and PCR-based PGT for mitochondrial DNA, our approach alleviates technical limitations by decreasing whole genome amplification artifacts by 68.4%, increasing breadth of coverage by at least 4-fold, and reducing wet-lab turn-around-time by ~2.5-fold. Importantly, this method enables trio-based PGT-A for aneuploidy origin, an approach we coin PGT-AO, detects translocation breakpoints, and nuclear and mitochondrial single nucleotide variants and indels in base-resolution.

High-throughput sequencing technologies have increasingly led to discovery of disease-causing genetic variants, primarily in postnatal multi-cell DNA samples. However, applying these technologies to preimplantation genetic testing (PGT) in nuclear or mitochondrial DNA from single or few-cells biopsied from in vitro fertilised (IVF) embryos is challenging. PGT aims to select IVF embryos without genetic abnormalities. Although genotyping-by-sequencing (GBS)-based haplotyping methods enabled PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A), they are labour intensive, only partially cover the genome and are troublesome for difficult loci and consanguineous couples. Here, we devised a simple, scalable and universal whole genome sequencing haplarithmisis-based approach enabling all forms of PGT in a single assay. In a comparison to state-of-the-art GBS-based PGT for nuclear DNA (37 embryos, 18 families, 25 indications), shallow sequencing-based PGT (10 embryos, 3 families), and PCR-based PGT for mitochondrial DNA (10 embryos, 2 families), our approach alleviates technical limitations by decreasing whole genome amplification artifacts by 68.4%, increasing breadth of coverage by 4-fold, and reducing wet-lab turn-around-time by 2.5-fold. Importantly, this method enables trio-based PGT-A for aneuploidy origin, an approach we coin PGT-AO, detects translocation breakpoints, and nuclear and mitochondrial single nucleotide variants and indels in base-resolution.

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.

Resolving the genetic architecture of painful neuropathy will lead to better disease management strategies. We aimed to develop a reliable method to re-sequence multiple genes in a large cohort of painful neuropathy patients at low cost. In this study, we compared sensitivity, specificity, targeting efficiency, performance and cost effectiveness of Molecular Inversion Probes-Next generation sequencing (MIPs-NGS) and TruSeq® Custom Amplicon-Next generation sequencing (TSCA-NGS). Capture probes were designed to target nine sodium channel genes (SCN3A, SCN8A-SCN11A, and SCN1B-SCN4B). One hundred sixty-six patients with diabetic and idiopathic neuropathy were tested by both methods, 70 patients were validated by Sanger sequencing. Sensitivity, specificity and performance of both techniques were comparable, and in agreement with Sanger sequencing. The average targeted regions coverage for MIPs-NGS was 97.3% versus 93.9% for TSCA-NGS. MIPs-NGS has a more versatile assay design and is more flexible than TSCA-NGS. The cost of MIPs-NGS is >5 times cheaper than TSCA-NGS when 500 or more samples are tested. In conclusion, MIPs-NGS is a reliable, flexible, and relatively inexpensive method to detect genetic variations in a large cohort of patients. In our centers, MIPs-NGS is currently implemented as a routine diagnostic tool for screening of sodium channel genes in painful neuropathy patients.