RESUMO
BACKGROUND: Liquid biopsy enables the non-invasive analysis of genetic tumor variants in circulating free DNA (cfDNA) in plasma. Accurate analytical validation of liquid biopsy NGS assays is required to detect variants with low variant allele frequencies (VAFs). METHODS: Six types of commercial cfDNA reference materials and 42 patient samples were analyzed using a duplex-sequencing-based liquid biopsy NGS assay. RESULTS: We comprehensively evaluated the similarity of commercial cfDNA reference materials to native cfDNA. We observed significant differences between the reference materials in terms of wet-lab and sequencing quality as well as background noise. No reference material resembled native cfDNA in all performance metrics investigated. Based on our results, we established guidelines for the selection of appropriate reference materials for the different steps in performance evaluation. The use of inappropriate materials and cutoffs could eventually lead to a lower sensitivity for variant detection. CONCLUSION: Careful consideration of commercial reference materials is required for performance evaluation of liquid biopsy NGS assays. While the similarity to native cfDNA aids in the development of experimental protocols, reference materials with well-defined variants are preferable for determining sensitivity and precision, which are essential for accurate clinical interpretation.
RESUMO
Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Neoplasias Hepáticas/genética , Progéria/genética , Idade de Início , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA/genética , Replicação do DNA/genética , Citometria de Fluxo , Imunofluorescência , Genes cdc/genética , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Peixe-Zebra/genéticaRESUMO
By using homozygosity mapping in a consanguineous Pakistani family, we detected linkage of nonsyndromic hearing loss to a 7.6 Mb region on chromosome 3q13.31-q21.1 within the previously reported DFNB42 locus. Subsequent candidate gene sequencing identified a homozygous nonsense mutation (c.1135G>T [p.Glu379X]) in ILDR1 as the cause of hearing impairment. By analyzing additional consanguineous families with homozygosity at this locus, we detected ILDR1 mutations in the affected individuals of 10 more families from Pakistan and Iran. The identified ILDR1 variants include missense, nonsense, frameshift, and splice-site mutations as well as a start codon mutation in the family that originally defined the DFNB42 locus. ILDR1 encodes the evolutionarily conserved immunoglobulin-like domain containing receptor 1, a putative transmembrane receptor of unknown function. In situ hybridization detected expression of Ildr1, the murine ortholog, early in development in the vestibule and in hair cells and supporting cells of the cochlea. Expression in hair cell- and supporting cell-containing neurosensory organs is conserved in the zebrafish, in which the ildr1 ortholog is prominently expressed in the developing ear and neuromasts of the lateral line. These data identify loss-of-function mutations of ILDR1, a gene with a conserved expression pattern pointing to a conserved function in hearing in vertebrates, as underlying nonsyndromic prelingual sensorineural hearing impairment.