Evaluation of in silico pathogenicity prediction tools for the classification of small in-frame indels.

BACKGROUND: The use of in silico pathogenicity predictions as evidence when interpreting genetic variants is widely accepted as part of standard variant classification guidelines. Although numerous algorithms have been developed and evaluated for classifying missense variants, in-frame insertions/deletions (indels) have been much less well studied. METHODS: We created a dataset of 3964 small (< 100 bp) indels predicted to result in in-frame amino acid insertions or deletions using data from gnomAD v3.1 (minor allele frequency of 1-5%), ClinVar and the Deciphering Developmental Disorders (DDD) study. We used this dataset to evaluate the performance of nine pathogenicity predictor tools: CADD, CAPICE, FATHMM-indel, MutPred-Indel, MutationTaster2021, PROVEAN, SIFT-indel, VEST-indel and VVP. RESULTS: Our dataset consisted of 2224 benign/likely benign and 1740 pathogenic/likely pathogenic variants from gnomAD (n = 809), ClinVar (n = 2882) and, DDD (n = 273). We were able to generate scores across all tools for 91% of the variants, with areas under the ROC curve (AUC) of 0.81-0.96 based on the published recommended thresholds. To avoid biases caused by inclusion of our dataset in the tools' training data, we also evaluated just DDD variants not present in either gnomAD or ClinVar (70 pathogenic and 81 benign). Using this subset, the AUC of all tools decreased substantially to 0.64-0.87. Several of the tools performed similarly however, VEST-indel had the highest AUCs of 0.93 (full dataset) and 0.87 (DDD subset). CONCLUSIONS: Algorithms designed for predicting the pathogenicity of in-frame indels perform well enough to aid clinical variant classification in a similar manner to missense prediction tools.

Systematic assessment of outcomes following a genetic diagnosis identified through a large-scale research study into developmental disorders.

PURPOSE: The clinical and psychosocial outcomes associated with receiving a genetic diagnosis for developmental disorders are wide-ranging but under-studied. We sought to investigate outcomes from a subset of families who received a diagnosis through the Deciphering Developmental Disorders (DDD) study. METHODS: Individuals recruited through the Peninsula Clinical Genetics Service who received a confirmed genetic diagnosis through the DDD study before August 2019 (n = 112) were included in a clinical audit. Families with no identified clinical outcomes (n = 16) were invited to participate in semistructured telephone interviews. RESULTS: Disease-specific treatment was identified for 7 probands (6%), while 48 probands (43%) were referred for further investigations or screening and 60 probands (54%) were recruited to further research. Just 5 families (4%) opted for prenatal testing in a subsequent pregnancy, reflecting the relatively advanced maternal age in our cohort, and 42 families (38%) were given disease-specific information or signposting to patient-specific resources such as support groups. Six interviews were performed (response rate = 47%) and thematic analysis identified four major themes: reaching a diagnosis, emotional impact, family implications, and practical issues. CONCLUSION: Our data demonstrate that receiving a genetic diagnosis has substantial positive medical and psychosocial outcomes for the majority of patients and their families.

Clinically-relevant postzygotic mosaicism in parents and children with developmental disorders in trio exome sequencing data.

Mosaic genetic variants can have major clinical impact. We systematically analyse trio exome sequence data from 4,293 probands from the DDD Study with severe developmental disorders for pathogenic postzygotic mosaicism (PZM) in the child or a clinically-unaffected parent, and use ultrahigh-depth sequencing to validate candidate mosaic variants. We observe that levels of mosaicism for small genetic variants are usually equivalent in both saliva and blood and ~3% of causative de novo mutations exhibit PZM; this is an important observation, as the sibling recurrence risk is extremely low. We identify parental PZM in 21 trios (0.5% of trios), resulting in a substantially increased sibling recurrence risk in future pregnancies. Together, these forms of mosaicism account for 40 (1%) diagnoses in our cohort. Likely child-PZM mutations occur equally on both parental haplotypes, and the penetrance of detectable mosaic pathogenic variants overall is likely to be less than half that of constitutive variants.

When genomic medicine reveals misattributed genetic relationships-the debate about disclosure revisited.

PURPOSE: Accidental discovery of misattributed parentage is an age-old problem in clinical medicine, but the ability to detect it routinely has increased recently as a result of high-throughput DNA sequencing technologies coupled with family sequencing studies. Problems arise at the clinical-research boundary, where policies and consent forms guaranteeing nondisclosure may conflict with standard clinical care. METHODS: To examine the challenges of managing misattributed parentage within hybrid translational research studies, we used a case study of a developmentally delayed child with a candidate variant found through a large-scale trio genome sequencing study in which data from unrelated samples were routinely excluded. RESULTS: We discuss whether genetic parentage should be explicitly confirmed during clinical validation, thus giving greater weight to the diagnosis according to American College of Medical Genetics and Genomics variant interpretation guidelines, and what tensions this approach would create. CONCLUSION: We recommend that the possibility of finding and disclosing misattributed parentage should be addressed during the consent or pretest counseling process, and that clinical relevance should determine whether or not to disclose results in the clinic. This proposition has implications for research governance, and implies that it may not always be possible to uphold nondisclosure commitments as investigations move from research to clinical care.

Risk prediction models: a framework for assessment.

BACKGROUND: Medical risk prediction models estimate the likelihood of future health-related events. Many make use of information derived from analysis of the genome. Models predict health outcomes such as cardiovascular disease, stroke and cancer, and for some conditions several models exist. Although risk models can help decision-making in clinical medicine and public health, they can also be harmful, for example, by misdirecting clinical effort away from those who are most likely to benefit towards people with less need, thus exacerbating health inequalities. DISCUSSION: Risk prediction models need careful assessment before implementation, but the current approach to their development, evaluation and implementation is inappropriate. As a result, some models are pressed into use before it is clear whether they are suitable, while in other cases there is confusion about which model to use. This paper proposes an approach to the appraisal of risk-scoring models, based on a conference of UK experts. SUMMARY: By specifying what needs to be known before a model can be judged suitable for translation from research into practice, we can ensure that useful models are taken up promptly, that less well-proven ones undergo further evaluation and that resources are not wasted on ineffective ones.

Non-invasive prenatal diagnosis using cell-free fetal DNA technology: applications and implications.

Cell-free fetal DNA and RNA circulating in maternal blood can be used for the early non-invasive prenatal diagnosis (NIPD) of an increasing number of genetic conditions, both for pregnancy management and to aid reproductive decision-making. Here we present a brief review of the scientific and clinical status of the technology, and an overview of key ethical, legal and social issues raised by the analysis of cell-free fetal DNA for NIPD. We suggest that the less invasive nature of the technology brings some distinctive issues into focus, such as the possibility of broader uptake of prenatal diagnosis and access to the technology directly by the consumer via the internet, which have not been emphasised in previous work in this area. We also revisit significant issues that are familiar from previous debates about prenatal testing. Since the technology seems to transect existing distinctions between screening and diagnostic tests, there are important implications for the form and process involved in obtaining informed consent or choice. This analysis forms part of the work undertaken by a multidisciplinary group of experts which made recommendations about the implementation of this technology within the UK National Health Service.

Short-lived carriage of foot-and-mouth disease virus in human nasal cavities after exposure to infected animals.

A quarantine period for potentially contaminated personnel can be used to reduce the risk of transfer of foot-and-mouth disease virus (FMDV) from infected to susceptible premises. This is set at 72 hours in the UK, on the basis of results from laboratory studies and field observations. Previous analysis of FMDV carriage within human nasal cavities has relied upon virus isolation by culture in susceptible cells. This study, involving 51 people, evaluated a PCR method, which detected viral genomic material within 35 nasal swabs taken from personnel after up to eight hours exposure to infected animals. Only one of 23 people who was PCR-positive immediately after exposure to FMDV-infected animals remained positive the following day, indicating a low risk of prolonged carriage of virus in the nasal cavities.

Biomarkers, dementia, and public health.

Public health is defined as the organized efforts of society to improve health. This is often framed in terms of prevention, with primary, secondary, and tertiary prevention representing, respectively, fundamental prevention through understanding of causation, to alteration of natural history, through understanding of pathophysiological mechanisms and palliation. Biomarkers play a role in all of these levels of prevention of dementias. The clearest application of biomarkers from a public health perspective is in the setting of screening. Screening has particular meaning for public health and includes early detection as a core element, coupled with treatments or preventative actions to reduce the burden of disease. Here, we will cover the range of evidence required if biomarkers are to play a part in population prevention of dementia, including scientific and technical aspects together with ethical, legal, and social considerations. Ensuring research activity that addresses these wider perspectives is essential.

Members of the hSWI/SNF chromatin remodeling complex associate with and are phosphorylated by protein kinase B/Akt.

In an adenosine triphosphate (ATP)-dependent process, the hSWI/SNF chromatin remodeling complex functions to alter chromatin structure, thereby regulating transcription factor access to DNA. In addition to interactions with transcription factors and recognition of acetylated histone residues, the chromatin remodeling activity of hSWI/SNF has also been shown to respond to a variety of cell signaling pathways. Our results demonstrate a novel interaction between the serine/threonine kinase Akt and members of the hSWI/SNF chromatin remodeling complex. Activation of Akt in HeLa cells resulted in its association with hSWI/SNF subunits: INI1, BAF155 and BAF170, as well as actin. BAF155 became preferentially recognized by an antibody that detects phosphorylated Akt substrates upon activation of Akt, suggesting that BAF155 may be an in vivo target for phosphorylation by Akt. Glutathione-S-transferase (GST) pulldown experiments demonstrated that INI1 and BAF155 were both capable of directly interacting with Akt. Finally, in vitro kinase assays provided additional evidence that BAF155 and potentially INI1 are substrates for Akt phosphorylation. These data provide the first evidence that Akt signaling may modulate function of the hSWI/SNF complex.

Heat- and lignosulfonate-treated canola meal as a source of ruminal undegradable protein for lactating dairy cows.

This experiment used 18 lactating Holstein cows in a 3 x 3 Latin square replicated 6 times to determine the effectiveness of processing with moist heat or moist heat combined with lignosulfonate (LSO3) for increasing the ruminal undegradable fraction of canola meal for use as a protein supplement for lactating dairy cows. Diets were formulated to be isonitrogenous and contained one of 3 forms of canola meal; untreated canola meal (UCM), heat-treated canola meal (HTCM) or heat-and LSO3-treated canola meal (LSO3CM). Total collection of urine and feces was taken from each cow during the last 5 d of each 42-d experimental period. Milk production was greater for cows fed the LSO3CM diet (36.6 kg/d) than for cows fed the UCM diet (34.8 kg/d) but did not differ from cows fed the HTCM diet (35.3 kg/d). Digestibility of crude protein was lower for cows supplemented with LSO3CM and they had reduced concentrations of ruminal ammonia N, blood urea N, and milk urea N compared with cows supplemented with UCM or HTCM. Dry matter intake and apparent digestibilities of neutral and acid detergent fiber were increased in cows fed the LSO3CM diet. Urinary N excretion (as % of N intake) was reduced in cows fed the LSO3CM diet. These results indicate that moist heat combined with LSO3 treatment of canola meal was effective in increasing the proportion of crude protein digested in the lower digestive tract of lactating cows and was therefore used more effectively as a source of protein than UCM or HTCM.

Vaccinia virus late transcription is activated in vitro by cellular heterogeneous nuclear ribonucleoproteins.

Vaccinia virus gene expression is temporally regulated, and three gene classes have been identified: early, intermediate, and late. Several virus-encoded proteins and an activity designated VLTF-X are required for maximum transcription in vitro of a template containing a late promoter. VLTF-X is present in both cytoplasmic and nuclear extracts prepared from uninfected mammalian cells and co-purifies with a late promoter DNA-binding activity. Here, extensive purification of VLTF-X has revealed that heterogeneous nuclear ribonucleoproteins A2/B1 and RBM3 co-purified with in vitro late transcription stimulation. Overexpression and purification of these proteins from Escherichia coli demonstrated that they both complemented for VLTF-X activity in in vitro transcription reactions. These studies identify two host cell factors potentially contributing to poxvirus replication in vivo.

A vaccinia virus late transcription factor with biochemical and molecular identity to a human cellular protein.

A factor designated VLTF-X is required to support vaccinia virus late transcription in vitro. It has been found that a late promoter DNA binding activity cochromatographs and cosediments with VLTF-X activity. Current experiments show that VLTF-X activity is present in a variety of uninfected mammalian cell types and is indistinguishable from that recovered from infected cells based upon several criteria. VLTF-X activity from both sources displays the same purification profile over phosphocellulose and DNA affinity resins and has the same sedimentation coefficient. In addition, the factors purified from both infected and uninfected cells form protein-DNA complexes of identical electrophoretic mobility in the presence of vaccinia virus late promoter-containing DNA. The affinity of these factors for the late promoter probes is identical and late promoter-specific based on competition experiments. Moreover, VLTF-X purified from both sources bound to late promoter-containing DNA in the presence or absence of MgCl2 and ATP and formed complexes resistant to heat inactivation. These experiments offer proof that vaccinia virus factor VLTF-X is a host cell protein that supports transcription of the viral late genes.

A vaccinia virus late transcription factor copurifies with a factor that binds to a viral late promoter and is complemented by extracts from uninfected HeLa cells.

We have previously described a vaccinia virus late transcription factor, VLTF-X, which we found to be present in cells at early and late times in infection. In this study, transcription complementation assays were used to demonstrate that VLTF-X activity is also present in virion extracts and in the cytoplasm of uninfected HeLa cells. Mobility shift assays performed on various VLTF-X preparations revealed that a late promoter DNA-binding activity cochromatographed and cosedimented with VLTF-X activity. Competition experiments demonstrated that this binding was specific for the late promoter region of the probe and that late transcription was dramatically reduced by an oligonucleotide that blocked factor-DNA complex formation but was only minimally affected by an oligonucleotide that did not inhibit complex formation. These results suggest that a cellular factor may participate in vaccinia virus late transcription. These findings also confirm the requirement for VLTF-X and distinguish it from any of the previously described vaccinia virus late transcription factors, which have all been mapped to the viral genome. Finally, these studies also suggest that the biochemical role for VLTF-X may be in late promoter recognition.

Establishment and molecular characterization of five cell lines derived from renal and extrarenal malignant rhabdoid tumors.

Malignant rhabdoid tumor (MRT) is a rare, enigmatic childhood cancer characterized by extreme aggressiveness and resistance to chemotherapy. To understand better the origin of the tumor and the mechanisms by which it develops and resists treatment, five cell lines were established from patients presenting with MRT (two renal and three extrarenal tumors). All of the cell lines display the light microscopic and ultrastructural features, as well as the variable immunohistochemical profile, characteristic of MRT. All are capable of forming tumors in nude mice. Three of the cell lines have detectable abnormalities of chromosome 22: one a t(22, 22) unbalanced translocation and two others a loss of heterozygosity of polymerase chain reaction-based microsatellite markers. Northern blot analysis showed that overexpression of the c-myc message was a consistent characteristic of the five MRTs evaluated. Although mutations of the p53 gene were not detectable by sequence analysis, all of the cell lines showed nuclear accumulation of the p53 protein by an immunocytochemical analysis in a minority of the cells. This result suggests that dysfunction in a p53-dependent apoptotic pathway might play a role in the multiple drug resistance phenotype of these tumors.

MDM2 amplification, P53 mutation, and accumulation of the P53 gene product in malignant fibrous histiocytoma.

Fifty-two cases of malignant fibrous histiocytoma (MFH) were evaluated for amplification of the MDM2 gene, mutation of the P53 gene, accumulation of the P53 gene product, and their relation to disease-free and overall survival. All tests were carried out on formalin-fixed, paraffin-embedded tissue samples. Amplification of the MDM2 gene was detected in 15 of 52 cases (29%). Six of 52 cases (12%) demonstrated abnormalities of the P53 gene. Sequence analysis detected point mutations in four cases and a 1-base pair deletion in one case, whereas differential polymerase chain reaction (dPCR) indicated that the P53 gene had been entirely deleted in one case. Eight of 52 cases (15%) demonstrated staining for the P53 protein in >10% of tumor cells. The presence of MDM2 amplification did not have a significant effect on either disease-free or overall survival. Patients with accumulation of the P53 gene product did not differ in disease-free or overall survival from patients without P53 accumulation. Survival also was not significantly different in patients with genetic aberration in P53. However, when the patients were stratified by histologic grade, the results indicated that patients with alterations in the P53 gene may have shorter overall survival.

The A2L intermediate gene product is required for in vitro transcription from a vaccinia virus late promoter.

Previously, the in vitro late transcription system of vaccinia virus was resolved into four components: the 17- and 30-kDa products of the A1L and G8R intermediate genes, respectively, the viral DNA-dependent RNA polymerase, and an unmapped factor sedimenting at 32 to 38 kDa. Another protein, the 26-kDa product of the A2L open reading frame was predicted to be a late transcription factor on the basis of a transient-expression assay but was not recognized as being necessary for transcriptional activity in vitro. We now report that both the unmapped factor and the 26-kDa protein are required for transcription from a vaccinia virus late promoter in vitro. Since the 26-kDa protein has now been shown to be a trans-activator of late transcription and it is the product of a known gene, we suggest that it be designated VLTF-3.

Discovery of the hemifumarate and (alpha-L-alanyloxy)methyl ether as prodrugs of an antirheumatic oxindole: prodrugs for the enolic OH group.

Ether, ester, and carbonate derivatives of the antirheumatic oxindole 1 were prepared and screened as potential prodrugs of 1. This effort led to the discovery of the (alpha-L-alanyloxy)-methyl ether and hemifumarate derivatives of 1 which deliver the drug efficiently into the circulation of test animals, are stable in the solid state, and possess good stability in solution at low pH as required to ensure gastric stability. Success in achieving acceptable bioavailabilities of 1 across species (rats, dogs, and monkeys) followed the inclusion of ionizable functionality within the promoiety to compensate for masking the polar enolic OH group of the free drug. However, the introduction of ionizable functionality was often associated with decreased stability, as demonstrated by the hemisuccinate, hemiadipate, hemisuberate, and alpha-amino ester derivatives of 1 which could not be isolated. A clear exception was the hemifumarate derivative of 1 which was not only isolable but actually more stable at neutral pH than the nonionizable ester analogues. The solution and solid state stability of the hemifumarate, together with its activity as a prodrug of 1, suggests that hemifumarate be considered as an alternative to hemisuccinate as a prodrug derivative for alcohols, particularly in situations where solution state stability is an issue.

The H4 subunit of vaccinia virus RNA polymerase is not required for transcription initiation at a viral late promoter.

Chromatography of RNA polymerase purified from vaccinia virions and from vaccinia virus-infected HeLa cells resulted in the separation of populations active for early and late transcription. An RNA polymerase population immunodepleted for the vaccinia virus H4 gene peptide could support late transcription.