Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments in NGS applied to the field of oncology. A number of clinical applications are reviewed, i.e., mutation detection in inherited cancer syndromes based on DNA-sequencing, detection of spliceogenic variants based on RNA-sequencing, DNA-sequencing to identify risk modifiers and application for pre-implantation genetic diagnosis, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy. Conclusive remarks, clinical limitations, implications and ethical considerations that relate to the different applications are provided.

Cardiovascular disease (CVD) is one of the biggest threats to public health worldwide. Identifying key genetic contributors to CVD enables clinicians to assess the most effective treatment course and prognosis, as well as potentially inform family members. This often involves either whole exome sequencing (WES) or targeted panel analysis of known pathogenic genes. In the future, tailored or personalized therapeutic strategies may be implemented, such as gene therapy. With the recent revolution in deep sequencing technologies, we know that up to 90% of the human genome is transcribed, despite only 2% of the 6 billion DNA bases coding for proteins. The long non-coding RNA (lncRNA) “genes” make up an important and significant fraction of this “dark matter” of the genome. We highlight how, despite lncRNA genes exceeding that of classical protein-coding genes by number, the “non-coding” human genome is neglected when looking for genetic components of disease. WES platforms and pathogenic gene panels still do not cover even characterized lncRNA genes that are functionally involved in the pathophysiology of CVD. We suggest that the importance of lncRNAs in disease causation and progression be taken as seriously as that of pathogenic protein variants and mutations, and that this is maybe a new area of attention for clinical geneticists.

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.

Primary mitochondrial disease (MD) is a group of rare genetic diseases reported to have a prevalence of 1:5000 and is currently without a cure. This group of diseases includes mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), maternally inherited diabetes and deafness (MIDD), Leber’s hereditary optic neuropathy (LHON), Leigh syndrome (LS), Kearns–Sayre syndrome (KSS), and myoclonic epilepsy and ragged-red fiber disease (MERRF). Additionally, secondary mitochondrial dysfunction has been implicated in the most common current causes of mortality and morbidity, including cardiovascular disease (CVD) and cancer. Identifying key genetic contributors to both MD and secondary mitochondrial dysfunction may guide clinicians to assess the most effective treatment course and prognosis, as well as informing family members of any hereditary risk of disease transmission. Identifying underlying genetic causes of primary and secondary MD involves either genome sequencing (GS) or small targeted panel analysis of known disease-causing nuclear- or mitochondrial genes coding for mitochondria-related proteins. Due to advances in GS, the importance of long non-coding RNA (lncRNA) as functional contributors to the pathophysiology of MD is being unveiled. A limited number of studies have thus far reported the importance of lncRNAs in relation to MD causation and progression, and we are entering a new area of attention for clinical geneticists in specific rare malignancies. This commentary provides an overview of what is known about the role of lncRNAs as genetic and molecular contributors to disease pathophysiology and highlights an unmet need for a deeper understanding of mitochondrial dysfunction in serious human disease burdens.

High mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (Tfam), a regulator of mtDNA replication, during early zebrafish development. Reduction ofTfamusing a splice-modifying morpholino (MO) resulted in a 42%±4% decrease in mtDNA copy number in embryos at 4 days post fertilization. Morphant embryos displayed abnormal development of the eye, brain, heart and muscle, as well as a 50%±11% decrease in ATP production. Transcriptome analysis revealed a decrease in protein-encoding transcripts from the heavy strand of the mtDNA. In addition, various RNA translation pathways were increased, indicating an upregulation of nuclear and mitochondria-related translation. The developmental defects observed were supported by a decreased expression of pathways related to eye development and haematopoiesis. The increase in mRNA translation might serve as a compensation mechanism, but appears insufficient during prolonged periods of mtDNA depletion, highlighting the importance of high mtDNA copy numbers for early development in zebrafish.SUMMARY STATEMENT: The first tuneable zebrafish model used to characterize the effect of a reduced mtDNA copy number and resulting OXPHOS deficiency on zebrafish embryonic development.