Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Integration of EpiSign, facial phenotyping, and likelihood ratio interpretation of clinical abnormalities in the re-classification of an ARID1B missense variant.

Heterozygous ARID1B variants result in Coffin-Siris syndrome. Features may include hypoplastic nails, slow growth, characteristic facial features, hypotonia, hypertrichosis, and sparse scalp hair. Most reported cases are due to ARID1B loss of function variants. We report a boy with developmental delay, feeding difficulties, aspiration, recurrent respiratory infections, slow growth, and hypotonia without a clinical diagnosis, where a previously unreported ARID1B missense variant was classified as a variant of uncertain significance. The pathogenicity of this variant was refined through combined methodologies including genome-wide methylation signature analysis (EpiSign), Machine Learning (ML) facial phenotyping, and LIRICAL. Trio exome sequencing and EpiSign were performed. ML facial phenotyping compared facial images using FaceMatch and GestaltMatcher to syndrome-specific libraries to prioritize the trio exome bioinformatic pipeline gene list output. Phenotype-driven variant prioritization was performed with LIRICAL. A de novo heterozygous missense variant, ARID1B p.(Tyr1268His), was reported as a variant of uncertain significance. The ACMG classification was refined to likely pathogenic by a supportive methylation signature, ML facial phenotyping, and prioritization through LIRICAL. The ARID1B genotype-phenotype has been expanded through an extended analysis of missense variation through genome-wide methylation signatures, ML facial phenotyping, and likelihood-ratio gene prioritization.

The Effects of Primary Unconjugated Bile Acids on Nanoencapsulated Pharmaceutical Formulation of Hydrophilic Drugs: Pharmacological Implications.

INTRODUCTION: In a recent study, in our laboratory, primary unconjugated bile acids, commonly found in humans, chenodeoxycholic acid (CDCA), have been shown to improve stability of nanoencapsulated lipophilic drugs and improve their release profile after oral administration likely via electrokinetic stabilisation. Hence, this study aimed to examine the effects of CDCA on exerting similar effects on hydrophilic drugs. METHODS: Various CDCA-based formulations were produced for the orally administered hydrophilic drug, metformin. Analyses of these formulations included electrokinetic potentials, topography, drug and CDCA formulation contents, nano size distribution, heat-induced deformation and outer-core expansion indices, release profiles, shell-resistance ratio, and thermal and chemical indices. With the drug's main target being pancreatic beta-cells, the formulations' effects on cell viability, functions and inflammatory profiles were also investigated. RESULTS AND CONCLUSIONS: CDCA-based metformin formulations exhibited improved stability and release profiles via thermal, chemical and electrokinetic effects, which were formulation-dependent suggesting potential applications of CDCA in the oral targeted delivery of hydrophilic drugs.

Bile acid permeation enhancement for inner ear cochlear drug pharmacological uptake: bio-nanotechnologies in chemotherapy-induced hearing loss.

Ototoxicity is the damage to inner ear sensory epithelia due to exposure to certain medications and chemicals. This occurs when toxins enter the tightly controlled inner ear environment inducing hair cell death, resulting in hearing loss. Recent studies have explored hydrogel-based bio-nanotechnologies and new drug delivery formulations to prevent drug-induced hearing loss, with much attention given to administration of antioxidant drugs. Bile acids have been recognized as promising excipients due to their biocompatibility and unique physiochemical properties. As yet bile acids have not been explored in improving drug delivery to the inner ear despite improving drug stability and delivery in other systems and demonstrating positive biological effects in their own right.

The Effects of Accelerated Temperature-Controlled Stability Systems on the Release Profile of Primary Bile Acid-Based Delivery Microcapsules.

INTRODUCTION: Bile acid-based drug encapsulation for oral delivery has been recently explored in our laboratory and has shown to be beneficial in terms of drug-targeted delivery and release profile, but stability at various temperatures has not previously been examined; hence, this is the aim of this study. METHODS: Various types of bile acid-based microcapsules containing the drug metformin were produced and tested for accelerated temperature-controlled profiles, as well as morphology, elemental composition, drug content, resilience, floatability, wettability and release profiles at various pH values. RESULTS: Accelerated temperature-controlled analysis showed negligible effects on morphology, size, or shape at very low temperatures (below 0 °C), while higher temperatures (above 25 °C) caused alterations. Drug contents, morphology and elemental composition remained similar, while wettability and the release profiles showed formulation-dependent effects. DISCUSSION AND CONCLUSION: Results suggest that bile acid-based microcapsules containing metformin are affected by temperature; hence, their shelf life is likely to be affected by storage temperature, all of which have a direct impact on drug release and stability profiles.

Pharmacological and Biological Study of Microencapsulated Probucol-Secondary Bile Acid in a Diseased Mouse Model.

Probucol (PB) is a highly lipophilic drug with potential protective effects on pancreatic ß-cells from inflammation and oxidation. PB has poor bioavailability and solubility, and despite many attempts, significant improvement in antidiabetic effects or absorption has yet to be discovered. Recently, the role of bile acids has been established in significant drug formulation stabilisation effects and as cell-penetrating agents. Promising results in pharmaceutical formulation studies on drug stability and release patterns when lithocholic acid (LCA) is conjugated with PB and sodium alginate (SA) have been demonstrated. Thus, this study aimed to develop and characterise PB microcapsules incorporating LCA and examine the biological effects of the microcapsules in vitro and in vivo. PB/LCA microcapsules were prepared using an encapsulation method, ionic gelation vibrational jet flow technology. LCA incorporation in PB microcapsules showed positive effects on ß-cells with improved insulin release, antioxidant activity, and PB intracellular uptake. Diabetic mice gavaged LCA-PB microcapsules showed a significant reduction in diabetes signs and symptoms, better survival rate, reduced blood glucose levels, and pro-inflammatory cytokines, with an increase PB level in blood and tissues suggesting a potential therapy for treating diabetes mellitus.

Foamy Virus Vectors Transduce Visceral Organs and Hippocampal Structures following In Vivo Delivery to Neonatal Mice.

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders.

Validation of a Chromosomal Microarray for Prenatal Diagnosis Using a Prospective Cohort of Pregnancies with Increased Risk for Chromosome Abnormalities.

AIM: Validation of a chromosomal microarray for improved prenatal diagnosis for chromosomal abnormalities among high-risk pregnancies. METHODS: A cohort of 213 pregnancies was investigated by chromosomal microarray and the results were compared with quantitative fluorescent polymerase chain reaction (QF-PCR), karyotype, and 850K single-nucleotide polymorphism microarray results. The detection limit of mosaicism was determined by assaying different trisomy mosaic constructs down to ∼12%. Imprecision estimates from replicates of mean log2 ratio values for a 200 kb deletion and 400 kb duplication were determined by evaluating the coefficient of variation (CV%). RESULTS: Excluding pregnancies with aneuploidy, the chromosomal microarray detected 19/213 (8.9%) pregnancies with copy number abnormalities. These were classified as pathogenic in 11/213 (5.2%) cases, as variants of uncertain significance in 4/213 (1.9%) cases, and as likely benign in 4/213 (1.9%) cases. In 15/213 (7.0%) pregnancies, these abnormalities were not detectable by karyotype. Importantly, 8/11 (72.7%) of the pathogenic abnormalities detected by chromosomal microarray were only detectable by this modality. There were no false-positive results and only eight false-negative results. The chromosomal microarray showed excellent sensitivity (96.2%) and specificity (100.0%). The lower detection limit for mosaicism was ∼12%. Imprecision for the 0.2 Mb deletion (11.6 CV%) and 0.4 Mb duplication (5.9 CV%) was very low. CONCLUSION: This chromosomal microarray showed excellent diagnostic performance with improved detection rates compared to karyotyping for prenatal diagnosis of clinically relevant fetal chromosomal abnormalities.

Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform.

Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC) protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.

Molecular genetic studies of complete hydatidiform moles.

Complete hydatidiform moles (CHM) are abnormal pregnancies with no fetal development resulting from having two paternal genomes with no maternal contribution. It is important to distinguish CHM from partial hydatidiform moles, and non-molar abortuses, due to the increased risk of gestational trophoblastic neoplasia. We evaluated a series of products of conception (POC) (n=643) investigated by genome-wide microarray comparative genomic hybridisation (CGH) with the aim of refining our strategy for the identification of complete moles. Among 32 suspected molar pregnancies investigated by STR genotyping to supplement microarray CGH testing, we found 31.3% (10/32) CHM; all identified among 3.6% (10/272) early first trimester POC. We suggest that when using microarray CGH that genotyping using targeted STR analysis should be performed for all POC referrals to aid in the identification of CHM.

Prenatal diagnosis of chromosomal mosaicism in over 1600 cases using array comparative genomic hybridization as a first line test.

OBJECTIVE: The aim of this study was to assess the detection of chromosomal mosaicism in chorionic villus (CVS) and amniotic fluid (AF) samples using array comparative genomic hybridization (aCGH) and quantitative fluorescent polymerase chain reaction. METHODS: All patients undergoing invasive prenatal testing by aCGH at a specialist prenatal screening service were included in the study. A total of 1609 samples (953 CVS and 656 AF) underwent quantitative fluorescent polymerase chain reaction and targeted aCGH without concurrent conventional G-banded karyotyping. RESULTS: Chromosomal mosaicism was detected in 20 of the 1609 cases (1.24%); of which 17 were derived from 953 CVS (1.78%), and three from 656 AF (0.46%). Mosaicism was observed at a level as low as 9%. Four cases were likely confined placental mosaicism, 12 were likely true fetal mosaicism, and four cases were unable to be classified into either group. CONCLUSIONS: This study demonstrates that the use of aCGH as a first line test is able to identify chromosomal mosaicism down to 9%, which is lower than the level reliably detected using standard cytogenetic analysis. aCGH avoids the disadvantages of culturing, which include culture bias, artifact, and culture failure.

Factors associated with the efficacy of a group intervention for anger in people with intellectual disabilities.

OBJECTIVES: There is a growing literature that suggests cognitive behavioural interventions aimed at reducing inappropriately expressed anger by people with intellectual disabilities are effective. However, interventions provide little information about which aspects of the individual or their treatment may be contributing to the overall efficacy of the approach. DESIGN: A group intervention is compared with a waiting list control. A cross-sectional regressional method was used to explore the relationship between change in a provocation inventory score over the course of an intervention and a number of other variables. METHODS: Data was collected from 50 participants who attended a series of groups with the aim of reducing aggressive behaviour. This was compared with a waiting list control of 36. Outcome was measured by a provocation inventory, which was administered pre-, post-group, and at follow-up. Other variables were also measured including, receptive vocabulary, age, gender, experience of primary therapist and whether staff accompanied participants to the group or not. RESULTS: A 2 x 2 split plot ANOVA identified a statistically significant treatment effect. However, analysis examining clinical significance was more equivocal with only 11 of the individuals in the intervention group showing reliable change as defined by Jacobson and Truax (1991). Regression analysis indicated that 17.5% of the variance in change of provocation inventory scores could be accounted for by the independent variables. Immediately on completion of the group, being accompanied by a member of staff and increased receptive language scores contributed significantly to the variance. CONCLUSIONS: A reduction in provocation inventory scores immediately after the group was more likely to occur if the participant was accompanied by a member of staff who knew them and if they had a higher score on the test of receptive vocabulary. This pattern changed with time. The implications of the results will be discussed and the relatively large amount of variance that is not accounted for will also be considered.

Range and prevalence of cardiac abnormalities in patients hospitalized in a medical ICU.

BACKGROUND: Patients hospitalized in medical ICUs (MICUs) with acute noncardiac illnesses have an undefined prevalence of underlying cardiovascular abnormalities. Because of the acuteness of illness, the need for frequent concurrent mechanical ventilation, and the nature of the underlying diseases, routine cardiac examination may be suboptimal for identifying concurrent cardiac abnormalities. PURPOSE: The purpose of this study was to utilize transthoracic echocardiography and Doppler echocardiography interrogation to identify the range and prevalence of occult cardiac abnormalities that may be present in patients admitted to an MICU. METHODS: Over a 12-month period, 500 consecutive patients who had been admitted to the MICU of a large university tertiary care center underwent complete two-dimensional echocardiography and Doppler scanning within 18 h of admission. The final study population comprised 467 patients. No study subject had been admitted to the MICU for a primary cardiac diagnosis. Cardiovascular abnormalities were prospectively defined, and all echocardiograms were interpreted independently by blinded observers. Both MICU and overall mortality rates as well as length of stay were compared to the presence or absence of cardiac abnormalities. RESULTS: One or more cardiac abnormalities was noted in 169 patients (36%). The average (+/-SD) age of patients in the study was 52 +/- 17 years (age range, 17 to 100 years), and the average age was 57 +/- 18 years (age range, 18 to 93 years) in patients with underlying cardiac abnormalities. A single cardiac abnormality was noted in 103 patients (22%), two cardiac abnormalities were noted in 34 patients (7.2%), and three or more cardiac abnormalities were noted in 32 patients (6.8%). Based on subsequent requests for cardiac diagnostic studies, 67 patients (14.3%) were clinically suspected of having significant cardiovascular abnormalities, 39 of whom (58%) had one or more cardiac abnormalities on seen on echocardiography. Cardiac abnormalities were unsuspected in 130 of 169 patients (77%) and were only noted at the time they underwent surveillance echocardiography. Although there was no correlation between the presence of cardiac abnormalities and mortality, both MICU and hospital length of stay were increased in patients with cardiac abnormalities. CONCLUSION: A significant proportion of patients admitted to an MICU with noncardiac illness have underlying cardiac abnormalities, which can be detected with surveillance echocardiography at the time of admission. Cardiac abnormalities were associated with an increased length of stay but not with increased mortality.