Delivering genome sequencing for rapid genetic diagnosis in critically ill children: parent and professional views, experiences and challenges

Rapid genomic sequencing (RGS) is increasingly being used in the care of critically ill children. Here we describe a qualitative study exploring parent and professional perspectives around the usefulness of this test, the potential for unintended harms and the challenges for delivering a wider clinical service. The Rapid Paediatric Sequencing (RaPS) study offered trio RGS for diagnosis of critically ill children with a likely monogenic disorder. Main and actionable secondary findings were reported. Semi-structured interviews were conducted with parents of children offered RGS (n = 11) and professionals (genetic clinicians, non-genetic clinicians, scientists and consenters) (n = 19) by telephone (parents n = 10/ professionals n = 1) or face-to-face (parents n = 1/professionals n = 18). We found that participants held largely positive views about RGS, describing clinical and emotional benefits from the opportunity to obtain a rapid diagnosis. Parental stress surrounding their child’s illness complicates decision making. Parental concerns are heightened when offered RGS and while waiting for results. The importance of multidisciplinary team working to enable efficient delivery of a rapid service was emphasised. Our findings give insight into the perceived value of RGS for critically ill children. Careful pre-test counselling is needed to support informed parental decision making. Many parents would benefit from additional support while waiting for results. Education of mainstream clinicians is required to facilitate clinical implementatio

Functional interaction between Sequestosome-1/p62 and Autophagy-Linked FYVE-containing protein WDFY3 in human osteoclasts

Peer reviewedPostprin

Lessons learnt from prenatal exome sequencing

BACKGROUND: Prenatal exome sequencing (ES) for monogenic disorders in fetuses with structural anomalies increases diagnostic yield. In England there is a national trio ES service delivered from two laboratories. To minimise incidental findings and reduce the number of variants investigated, analysis uses a panel of 1205 genes where pathogenic variants may cause abnormalities presenting prenatally. Here we review our laboratory's early experience developing and delivering ES to identify challenges in interpretation and reporting and inform service development. METHODS: A retrospective laboratory records review from 01.04.2020 to 31.05.2021. RESULTS: Twenty-four of 116 completed cases were identified as challenging including 13 resulting in difficulties in analysis and reporting, nine where trio inheritance filtering would have missed the diagnosis, and two with no prenatal diagnosis; one due to inadequate pipeline sensitivity, the other because the gene was not on the panel. Two cases with copy number variants identified were not detectable by microarray. CONCLUSIONS: Variant interpretation requires close communication between referring clinicians, with occasional additional examination of the fetus or parents and communication of evolving phenotypes. Inheritance filtering misses ∼5% of diagnoses. Panel analysis reduces but does not exclude incidental findings. Regular review of published literature is required to identify new reports that may aid classification

Physical Activity and Sedentary Behaviour Clustering: Segmentation to Optimise Active Lifestyles.

Background: Increasingly the health impacts of physical inactivity are being distinguished from those of sedentary behaviour. Nevertheless, deleterious health prognoses occur when these behaviours combine, making it a Public Health priority to establish the numbers and salient identifying factors of people who live with this injurious combination. Method: Using an observational between-subjects design, a non-probability sample of 22,836 participants provided data on total daily activity. A 2-step hierarchical cluster analysis identified the optimal number of clusters and the subset of distinguishing variables. Univariate analyses assessed significant cluster differences. Results: High levels of sitting clustered with low physical activity. The ‘Ambulatory & Active’ cluster (n=6,254) sat for 2.5 to 5 h d-1 and were highly active. They were significantly younger, included a greater proportion of males and reported low Indices of Multiple Deprivation compared to other clusters. Conversely, the ‘Sedentary & Low Active’ cluster (n=6,286) achieved ≤60 MET.min.wk-1 of physical activity and sat for ≥8 h d-1. They were the oldest cluster, housed the largest proportion of females and reported moderate Indices of Multiple Deprivation. Conclusions: Public Health systems may benefit from developing policy and interventions that do more to limit sedentary behaviour and encourage light intensity activity in its place

Safety and feasibility of sublingual microcirculation assessment in the emergency department for civilian and military patients with traumatic haemorrhagic shock: a prospective cohort study

OBJECTIVES: Sublingual microcirculatory monitoring for traumatic haemorrhagic shock (THS) may predict clinical outcomes better than traditional blood pressure and cardiac output, but is not usually performed until the patient reaches the intensive care unit (ICU), missing earlier data of potential importance. This pilot study assessed for the first time the feasibility and safety of sublingual video-microscopy for THS in the emergency department (ED), and whether it yields useable data for analysis. SETTING: A safety and feasibility assessment was undertaken as part of the prospective observational MICROSHOCK study; sublingual video-microscopy was performed at the UK-led Role 3 medical facility at Camp Bastion, Afghanistan, and in the ED in 3 UK Major Trauma Centres. PARTICIPANTS: There were 15 casualties (2 military, 13 civilian) who presented with traumatic haemorrhagic shock with a median injury severity score of 26. The median age was 41; the majority (n=12) were male. The most common injury mechanism was road traffic accident. PRIMARY AND SECONDARY OUTCOME MEASURES: Safety and feasibility were the primary outcomes, as measured by lack of adverse events or clinical interruptions, and successful acquisition and storage of data. The secondary outcome was the quality of acquired video clips according to validated criteria, in order to determine whether useful data could be obtained in this emergency context. RESULTS: Video-microscopy was successfully performed and stored for analysis for all patients, yielding 161 video clips. There were no adverse events or episodes where clinical management was affected or interrupted. There were 104 (64.6%) video clips from 14 patients of sufficient quality for analysis. CONCLUSIONS: Early sublingual microcirculatory monitoring in the ED for patients with THS is safe and feasible, even in a deployed military setting, and yields videos of satisfactory quality in a high proportion of cases. Further investigations of early microcirculatory behaviour in this context are warranted. TRIAL REGISTRATION NUMBER: NCT02111109

Fetal central nervous system anomalies: When should we offer exome sequencing?

OBJECTIVE: To investigate the detection of pathogenic variants using exome sequencing in an international cohort of fetuses with central nervous system (CNS) anomalies. METHODS: We reviewed trio exome sequencing (ES) results for two previously reported unselected cohorts (Prenatal Assessment of Genomes and Exomes (PAGE) and CUIMC) to identify fetuses with CNS anomalies with unremarkable karyotypes and chromosomal microarrays. Variants were classified according to ACMG guidelines and association of pathogenic variants with specific types of CNS anomalies explored. RESULTS: ES was performed in 268 pregnancies with a CNS anomaly identified using prenatal ultrasound . Of those with an isolated, single, CNS anomaly, 7/97 (7.2%) had a likely pathogenic/pathogenic (LP/P) variant. This includes 3/23 (13%) fetuses with isolated mild ventriculomegaly and 3/10 (30%) fetuses with isolated agenesis of the corpus callosum. Where there were multiple anomalies within the CNS, 12/63 (19%) had LP/P variants. Of the 108 cases with CNS and other organ system anomalies, 18 (16.7%) had LP/P findings. CONCLUSION: ES is an important tool in the prenatal evaluation of fetuses with any CNS anomaly. The rate of LP/P variants tends to be highest in fetuses with multiple CNS anomalies and multisystem anomalies, however, ES may also be of benefit for isolated CNS anomalies

Noninvasive Prenatal Diagnosis for Cystic Fibrosis: Implementation, Uptake, Outcome, and Implications

BACKGROUND: Noninvasive prenatal diagnosis (NIPD) for monogenic disorders has a high uptake by families. Since 2013, our accredited public health service laboratory has offered NIPD for monogenic disorders, predominantly for de novo or paternally dominantly inherited mutations. Here we describe the extension of this service to include definitive NIPD for a recessive condition, cystic fibrosis (CF). // METHODS: Definitive NIPD for CF was developed using next-generation sequencing. Validation was performed on 13 cases from 10 families before implementation. All cases referred for CF NIPD were reviewed to determine turnaround times, genotyping results, and pregnancy outcomes. // RESULTS: Of 38 referrals, 36 received a result with a mean turnaround of 5.75 days (range, 3-11 days). Nine cases were initially inconclusive, with 3 reported unaffected because the low-risk paternal allele was inherited and 4 cases in which the high-risk paternal allele was inherited, receiving conclusive results following repeat testing. One case was inconclusive owing to a paternal recombination around the mutation site, and one case was uninformative because of no heterozygosity. Before 2016, 3 invasive referrals for CF were received annually compared with 38 for NIPD in the 24 months since offering a definitive NIPD service. // CONCLUSIONS: Timely and accurate NIPD for definitive prenatal diagnosis of CF is possible in a public health service laboratory. The method detects recombinations, and the service is well-received as evidenced by the significant increase in referrals. The bioinformatic approach is gene agnostic and will be used to expand the range of conditions tested for

Signal peptide mutations in RANK prevent downstream activation of NF-κB

Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF-κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF-κB occurred in HEK293 cells overexpressing wild-type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild-type RANK demonstrated ligand-dependent activation of NF-κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild-type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins. © 2011 American Society for Bone and Mineral Researc