Factors associated with the time to complete clinical exome sequencing in a pediatric patient population.

PURPOSE: Exome sequencing (ES) is becoming increasingly important for diagnosing rare genetic disorders. Patients and clinicians face several barriers when attempting to obtain ES. This study is aimed to describe factors associated with a longer time interval between provider recommendation of testing and sample collection for ES. METHODS: A retrospective chart review was conducted for insurance-authorized, completed pediatric ES in which initial requests were reviewed by Stanford's Genetic Testing Optimization Service between November 2018 and December 2019. Regression analysis was used to determine the association between the geocoded median household income and 3 different time point intervals defined as time to test, insurance decision, and scheduling/consent. RESULTS: Of the 281 charts reviewed, 115 cases were included in the final cohort. The average time from provider preauthorization request to sample collection took 104.4 days, and income was negatively correlated with the length of the insurance decision interval. CONCLUSION: Pediatric patients undergo a lengthy, uncertain process when attempting to obtain ES, some of which is associated with income. More research and clinician interventions are required to clarify specific socioeconomic factors that influence the ability to obtain timely ES and develop optimal protocols.

C-NHEJ without indels is robust and requires synergistic function of distinct XLF domains.

To investigate the fidelity of canonical non-homologous end joining (C-NHEJ), we developed an assay to detect EJ between distal ends of two Cas9-induced chromosomal breaks that are joined without causing insertion/deletion mutations (indels). Here we find that such EJ requires several core C-NHEJ factors, including XLF. Using variants of this assay, we find that C-NHEJ is required for EJ events that use 1-2, but not ≥3, nucleotides of terminal microhomology. We also investigated XLF residues required for EJ without indels, finding that one of two binding domains is essential (L115 or C-terminal lysines that bind XRCC4 and KU/DNA, respectively), and that disruption of one of these domains sensitizes XLF to mutations that affect its dimer interface, which we examined with molecular dynamic simulations. Thus, C-NHEJ, including synergistic function of distinct XLF domains, is required for EJ of chromosomal breaks without indels.

PRPF8 is important for BRCA1-mediated homologous recombination.

Disruption of RNA splicing causes genome instability, which could contribute to cancer etiology. Furthermore, RNA splicing is an emerging anti-cancer target. Thus, we have evaluated the influence of the spliceosome factor PRPF8 and the splicing inhibitor Pladienolide B (PlaB) on homologous recombination (HR). We find that PRPF8 depletion and PlaB treatment cause a specific defect in homology-directed repair (HDR), and single strand annealing (SSA), which share end resection as a common intermediate, and BRCA1 as a required factor. Furthermore, PRPF8 depletion and PlaB treatment cause reduced end resection detected as chromatin-bound RPA, BRCA1 foci in response to damage, and histone acetylation marks that are associated with BRCA1-mediated HR. We also identified distinctions between PlaB and PRPF8 depletion, in that PlaB also reduces 53BP1 foci, and BRCA1 expression. Furthermore loss of 53BP1, which rescues SSA in BRCA1 depleted cells, and partially rescues SSA in PRPF8 depleted cells, has no effect on SSA in PlaB treated cells. Finally, while PRPF8 depletion has no obvious effect on the integrity of interchromatin granules, PlaB disrupts these structures. These findings indicate that PRPF8 is important for BRCA1-mediated HR, whereas PlaB also has a more general effect on the DNA damage response and nuclear organization.

Contribution of canonical nonhomologous end joining to chromosomal rearrangements is enhanced by ATM kinase deficiency.

A likely mechanism of chromosomal rearrangement formation involves joining the ends from two different chromosomal double-strand breaks (DSBs). These events could potentially be mediated by either of two end-joining (EJ) repair pathways [canonical nonhomologous end joining (C-NHEJ) or alternative end joining (ALT-EJ)], which cause distinct rearrangement junction patterns. The relative role of these EJ pathways during rearrangement formation has remained controversial. Along these lines, we have tested whether the DNA damage response mediated by the Ataxia Telangiectasia Mutated (ATM) kinase may affect the relative influence of C-NHEJ vs. ALT-EJ on rearrangement formation. We developed a reporter in mouse cells for a 0.4-Mbp deletion rearrangement that is formed by EJ between two DSBs induced by the Cas9 endonuclease. We found that disruption of the ATM kinase causes an increase in the frequency of the rearrangement as well as a shift toward rearrangement junctions that show hallmarks of C-NHEJ. Furthermore, ATM suppresses rearrangement formation in an experimental condition, in which C-NHEJ is the predominant EJ repair event (i.e., expression of the 3' exonuclease Trex2). Finally, several C-NHEJ factors are required for the increase in rearrangement frequency caused by inhibition of the ATM kinase. We also examined ATM effectors and found that H2AX shows a similar influence as ATM, whereas the influence of ATM on this rearrangement seems independent of 53BP1. We suggest that the contribution of the C-NHEJ pathway to the formation of a 0.4-Mbp deletion rearrangement is enhanced in ATM-deficient cells.

Clinical phenotype and allergen sensitization in the first 2 years as predictors of atopic disorders at age 5 years.

INTRODUCTION: From a birth cohort of at-risk Asian infants, we prospectively investigated the role of early onset allergen sensitization and clinical phenotypes as risk factors for atopic disorders at the age of 5 years. METHODS AND MATERIALS: The study recruited 253 families with a history of allergic disease in a first degree relative from an antenatal clinic in Singapore. The children were followed prospectively to assess clinical outcomes and skin prick test was performed at 2 and 5 years of age. RESULTS: Allergen sensitization (food and/or house dust mites) alone at 2 years of age was not associated with increased risk of wheeze and eczema at 5 years. However, the clinical phenotype (eczema and wheeze) with or without the presence of concomitant allergen sensitization at 2 years increased this risk. For eczema, eczema alone at year 2 increased the risk of eczema at year 5 (adjOR = 7.1; 95 % CI: 1.8-27.8) and this was further increased by the presence of allergen sensitization (adjOR = 25.4; 95 % CI: 4.7-138.5) and the concomitant presence of both wheeze and allergen sensitization (adjOR = 64.9; 95 % CI: 4.7-900.0). For wheeze, wheeze alone at 2 years (adjOR = 4.5; 95 % CI: 1.4 -14.8), and wheeze with concomitant allergen sensitization and eczema (adjOR = 13.9; 95 % CI: 1.2-168.5) increased the risk of wheeze at 5 years. The exception was rhinitis, where allergen sensitization alone at 2 years (adjOR = 5.6; 95 % CI: 1.1-29.2) increased the risk of rhinitis at 5 years. Early onset of eczema at 2 years also increased the risk of rhinitis (adjOR = 6.8; 95 % CI: 2.0-23.1). CONCLUSION: In this Asian birth cohort, the clinical phenotype (eczema and wheeze) with or without concomitant allergen sensitization in the first 2 years of life were strong predictors of atopic disorders at 5 years.