Envisioning the development of a CRISPR-Cas mediated base editing strategy for a patient with a novel pathogenic CRB1 single nucleotide variant.

BACKGROUND: Inherited retinal degeneration (IRD) associated with mutations in the Crumbs homolog 1 (CRB1) gene is associated with a severe, early-onset retinal degeneration for which no therapy currently exists. Base editing, with its capability to precisely catalyse permanent nucleobase conversion in a programmable manner, represents a novel therapeutic approach to targeting this autosomal recessive IRD, for which a gene supplementation is challenging due to the need to target three different retinal CRB1 isoforms. PURPOSE: To report and classify a novel CRB1 variant and envision a possible therapeutic approach in form of base editing. METHODS: Case report. RESULTS: A 16-year-old male patient with a clinical diagnosis of early-onset retinitis pigmentosa (RP) and characteristic clinical findings of retinal thickening and coarse lamination was seen at the Oxford Eye Hospital. He was found to be compound heterozygous for two CRB1 variants: a novel pathogenic nonsense variant in exon 9, c.2885T>A (p.Leu962Ter), and a likely pathogenic missense change in exon 6, c.2056C>T (p.Arg686Cys). While a base editing strategy for c.2885T>A would encompass a CRISPR-pass mediated "read-through" of the premature stop codon, the resulting missense changes were predicted to be "possibly damaging" in in-silico analysis. On the other hand, the transversion missense change, c.2056C>T, is amenable to transition editing with an adenine base editor (ABE) fused to a SaCas9-KKH with a negligible chance of bystander edits due to an absence of additional Adenines (As) in the editing window. CONCLUSIONS: This case report records a novel pathogenic nonsense variant in CRB1 and gives an example of thinking about a base editing strategy for a patient compound heterozygous for CRB1 variants.

A novel homozygous c.67C>T variant in retinol binding protein 4 (RBP4) associated with retinitis pigmentosa and childhood acne vulgaris.

BACKGROUND: The retinol binding protein 4 (RBP4) is essential in delivering retinol to the retinal pigment epithelium and normal functioning of the visual cycle. Homozygous mutations in the RBP4 gene lead to severe retinitis pigmentosa that is phenotypically indistinguishable from retinitis pigmentosa caused by other recessive mutations. METHODS: Case Report. PURPOSE: To report a novel homozygous RBP4 c.67 C > T variant in a case of retinitis pigmentosa associated with severe childhood acne vulgaris. RESULTS: A 49-year old Caucasian man with a family history of retinitis pigmentosa, presented with low vision and night blindness from early childhood. Fundus examination showed findings typical of recessive retinitis pigmentosa. Next generation sequencing analysis revealed a novel homozygous RBP4 c.67 C > T variant. Examination of patient's back showed widespread scaring and hyperpigmentation secondary to severe childhood-onset acne vulgaris. Patient's affected brother, positive for the same homozygous variant, also had a history of severe acne vulgaris whereas the unaffected brother did not, confirming that mutations in RBP4 segregated with the acne vulgaris phenotype in this family. CONCLUSIONS: We describe a case of retinitis pigmentosa associated with acne vulgaris and highlight the role of this systemic manifestation of retinol deficiency in confirming pathogenicity of the novel variant. Given the small size of the genomic RBP4 DNA (0.6kb), gene therapy using an adeno-associated viral vector with subretinal delivery has great potential to treat this severe childhood-onset blinding retinal disease. In addition, ubiquitous expression of RBP4 supports the development of in vitro functional assays to test the vector potency for clinical use.

Novel non-contiguous exon duplication in choroideremia.

The importance of establishing a genetic diagnosis in patients with a choroideremia phenotype has been underscored by the advent of gene replacement therapy for this condition. Here, we describe a complex imbalance at the CHM locus in a male patient with classical disease. At the DNA level, this imbalance consists of 2 non-contiguous duplications (exons 1-2 and 9-12). Further characterization suggests the generation of 2 independent CHM transcriptional units, one of which may produce a deleted form of the Rab escort protein 1 protein. Expression of such a type of aberrant protein in photoreceptors may have important implications when considering gene therapy for this disorder.

Relationship of pressure to be thin with gains in body weight and fat mass in adolescents.

BACKGROUND: Sociocultural pressure to be thin is commonly reported by adolescents; yet, to what extent such pressure is associated with weight gain has not been evaluated longitudinally. OBJECTIVE: Examine whether pressure to be thin was positively associated with weight and fat gain in adolescents. METHODS: Participants were 196 healthy adolescent (age 15 ± 1 years old) girls (65%) and boys of varying weights (BMI 25 ± 7 kg/m2 ) studied at baseline and 1-year follow-up. At baseline, adolescents and their mothers reported pressure to be thin by questionnaire. At baseline and follow-up, BMI was calculated, and fat mass was assessed with air displacement plethysmography. Multiple regression was used to examine associations between baseline pressure to be thin and 1-year changes in BMI and fat mass. RESULTS: Accounting for multiple covariates, including baseline BMI or fat, adolescent-reported pressure from parents and peers and mother-reported pressure toward their teen were associated with greater gains in either adolescent BMI or fat (ps < .05). Adolescent weight status was a moderator of multiple effects (ps < .05). CONCLUSIONS: Parental and peer pressure to be thin were associated with increases in BMI and fat mass during adolescence, particularly in heavier adolescents. Further research is necessary to clarify how this association operates reciprocally and to identify underlying explanatory mechanisms.

Established donepezil treatment modulates task relevant regional brain activation in early Alzheimer's disease.

Neuroimaging studies of cholinesterase inhibitor (ChEI) treatment in Alzheimer's disease (AD) have shown the different short and long term actions of ChEIs. fMRI studies of the ChEI donepezil have focused on its short to medium term action without exploring the effects of established treatment. In this exploratory study the effect of 20 weeks donepezil treatment on regional brain activity was measured with fMRI in patients with mild AD. Twelve patients with probable AD and nine age-matched controls were assessed with a Pyramids and Palm Trees semantic association fMRI paradigm and an n-back working memory fMRI paradigm. In the patient group only, the assessment was repeated after 20 weeks of treatment. After treatment, differences from normal healthy elderly became more pronounced. There was also a spread of deactivation which at retest was detectable in task relevant areas. Behaviourally, however, there were no significant differences between group baseline and retest scores, with a range of performance probably reflecting variation in drug efficacy across patients. Parametric analyses established that increased behavioural scores at retest correlated significantly with higher activation levels in non task relevant areas. Behavioural stability with donepezil treatment was not paralleled by the pattern of improved task specific brain activation reported in similar studies of other ChEIs. This is arguably related to the different mechanisms of action of the ChEIs and might be a clinical correlate of the reported synaptic upregulation following long term donepezil treatment.

Advanced applications of 3-dimensional echocardiography.

Over the last few decades, advancements in ultrasound, electronic and computing technologies have permitted current second generation 3-dimensional (3D) echocardiography to display on-line 3D rendered images of the heart. Since various studies demonstrated its superiority over 2-dimensional echocardiography, there is growing enthusiasm to embrace this new 3D echocardiographic technology. With its increasing widespread clinical availability, 3D echocardiography is getting closer to routine clinical use. However, as with any new emerging technologies, clinical applications of 3D echocardiography should be based on current evidence. This review will focus on the evidence from clinical studies that form the scientific basis for the advanced applications of 3D echocardiography, from cardiac chamber volume assessments, left ventricular dyssynchrony assessments, quantifications of valvular abnormalities, to the role of 3D echocardiography during cardiac interventions.

Left atrial appendage thrombus in a patient in sinus rhythm with endocarditis and a severe aortic valve insufficiency.

A left atrial thrombus is most often associated with atrial fibrillation and/or rheumatic mitral stenosis. It is very infrequently detected in the presence of sinus rhythm. The present report describes the case of a 66-year-old woman who presented with a stroke and was subsequently found to have two potential sources of embolization, including a vegetation on the native aortic valve, with associated severe aortic insufficiency, and a left atrial appendage thrombus despite being in sinus rhythm. To the authors' knowledge, the present report is the first to describe a left atrial thrombus in sinus rhythm associated with aortic valve endocarditis.

Structural and chemical changes in ultra-high-molecular-weight polyethylene due to gamma radiation-induced crosslinking and annealing in air.

Ultra-High-Molecular-Weight-Polyethylene (UHMWPE) is the material of choice for one of the articulating surfaces in many total joint replacements, notably hip and knee prostheses. The various methods used by the orthopaedic biomaterials industry to sterilize and anneal UHMWPE components, and the resulting oxidation and crosslinking, affect the mechanical wear resistance properties in ways still unknown at the microscopic and molecular levels. Transmission electron microscopy and chemical pyrolysis were used to quantify crosslinking induced by gamma irradiation and annealing in air. Changes in lamellar stacking and the amount of crosslinking suggest two types of crosslinking: relatively unstable crosslinks in the amorphous region initially resulting from gamma irradiation which are later replaced by more thermally stable crosslinks resulting from rearrangements at the annealing temperature. Lamellar mobility, the ability of crystalline lamellae to flow in the material, is enhanced during the transition from one type of bond to the other, and this appears to optimize near eight hours of annealing time. Results from decomposition and percent crystallinity measurements provide further support for this theory.

The use of viral vectors to produce hepatitis B virus core particles in plants.

The expression and assembly of the hepatitis B virus (HBV) nucleocapsid protein (HBcAg) were investigated in plants using viral vectors. Constructs based on either Potato virus X (PVX) or Cowpea mosaic virus (CPMV) containing the sequence of HBcAg were able to infect the appropriate host plants and remained genetically stable during infection. Analysis of HBcAg expression revealed that the protein can self-assemble into core-like particles and that the assembled material could be partially purified by differential centrifugation. Thus, the use of viral vectors can be considered a practical method for rapid production of assembled HBcAg particles in plants. This approach provides a means whereby a variety of chimaeric particles can be assessed quickly and cheaply for various diagnostic and vaccine applications.

Cochlear implantation and management of chronic suppurative otitis media: single stage procedure?

In a series of 360 patients who underwent cochlear implantation at our center, four patients (five procedures) had cochlear implantation with obliteration of the mastoid cavity and management of cholesteatoma as a single-staged procedure. Three patients were bilaterally deaf secondary to CSOM and had bilateral mastoid cavities, and in one patient congenital cholesteatoma was identified during cochlear implantation. A mastoidectomy or revision mastoidectomy with obliteration of the mastoid cavity and cochlear implantation was performed as a single stage procedure. Cholesteatoma reoccurred in one patient 9 years after cochlear implantation. Surgical procedures, complications, follow-up and outcomes are discussed.

Short-term longitudinal evaluation of cerebral blood flow in mild Alzheimer's disease.

The aim of this study was to estabilish whether subtle changes in regional cerebral blood flow (rCBF) can be detected in patients with mild Alzheimer's disease (AD) over a period as short as three months. Aprospective evaluation of rCBF changes with HMPAO SPECT at baseline and after 3 and 6 months was carried out. Standard clinical dementia tests were performed in parallel. The study enrolled 13 patients with mild probable AD from an outpatient memory clinic. SPECT data collected at baseline and after three months were compared using Statistical Parametric Mapping (SPM). Significant clusters of rCBF decrement in the medial part of the temporal lobe in both hemispheres, in the orbital part of the right frontal lobe and in the inferior part of the right parietal lobe were observed. No significant differences were found using a region of interest (ROI) analysis. After six months SPM analysis showed larger clusters of significant decrement in the same areas. ROI analysis was now sensitive to the rCBF changes and significant differences were shown in all brain regions except the temporal lobes. SPM analysis of SPECT data can detect significant changes in rCBF in mild AD over a short time interval. This method might be of potential advantage for the early diagnosis of AD and for its differentiation from stable cognitive impairments.

Preservation of golf skills in a case of severe left lobar frontotemporal degeneration.

The authors report the longitudinal study of a 53-year-old man with severe lobar atrophy confined to the left frontal and temporal lobes, including the left hippocampus, but sparing other cortical regions. He experienced profound cognitive deterioration, sparing only visuospatial memory. Despite these deficits, he could play golf at a high level of competence, following rules and etiquette as well as monitoring the ongoing game. The patient's golf performance may have been supported by residual visuospatial declarative memory and complex flexible implicit memory programs.

Quality of spelling breakdown in Alzheimer's disease is independent of disease progression.

There is a convergence of evidence from recent studies that spelling is impaired in Alzheimer's Disease (AD) relative to normal aging. However, the literature is more divided over whether there is a logical pattern in spelling deterioration with disease progression. The present study reviewed performance of participants with mild AD, moderate AD, and control participants on a written and oral spelling task. Results suggest that spelling in individuals with AD is impaired relative to controls. Comparison between those with mild AD and moderate AD failed to find evidence of a logical pattern of deterioration. We further examined the effects of word characteristics on spelling performance. Although increasing in quantity with disease progression, analysis of quality of spelling errors reveal a multi-componential disruption of spelling ability, which is independent of disease progression.

HMPAO SPECT imaging of Alzheimer's disease patients with similar content-specific autobiographic delusion: comparison using statistical parametric mapping.

UNLABELLED: Delusional behavior and thinking are common symptoms in Alzheimer's disease (AD). In the past, these delusions have been considered to be psychotic complications of global neurologic dysfunction. Recently, authors have suggested that content-specific delusions in AD are associated with discrete regional abnormalities of the right hemisphere. METHOD: This study compared 99mTc-hexamethyl propyleneamine oxime (HMPAO) SPECT images of a group of AD patients with a similar autobiographic delusion with a group of AD patients without delusions and a group of AD patients with a range of delusions but without autobiographic content. The reconstructed SPECT data were compared using a statistical parametric mapping technique. RESULTS: The autobiographic AD group had a significant area of hypoperfusion in the right frontal lobe when compared with the 2 other groups. The area of hypoperfusion included parts of Brodmann's areas 9 and 10. Region 9 has been identified previously as having a role in episodic memory retrieval. CONCLUSION: This result suggests that autobiographic delusions in AD may have an identifiable neuropsychologic mechanism and that it may be possible to identify an organic cause in some patients using 99mTc-HMPAO SPECT.

Association of phosphatidylinositol 3-kinase with nuclear transcription sites in higher plants.

The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti-bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants.

Changes in the rCBF images of patients with Alzheimer's disease receiving Donepezil therapy.

Alzheimer's disease is associated with a loss in presynaptic cholinergic function. It has been suggested that cholinergic inhibitors such as donepezil hydrochloride (Donepezil) could restore this function and improve some of the symptoms of Alzheimer's disease. Previous work has shown that Donepezil improves cognitive and global function in patients with mild to moderate Alzheimer's disease. This study reviewed retrospectively 12 patients who had previously had a 99Tcm-hexamethylpropylene amine oxime (99Tcm-HMPAO) single photon emission tomography (SPET) regional cerebral blood flow (rCBF) examination and had gone on to receive Donepezil therapy. These patients were recalled for a further 99Tcm-HMPAO SPET rCBF examination and the image data sets were compared. The results showed an overall increase in global cerebral blood flow (P = 0.04) averaged over the group with a percentage change in blood flow ranging from -1.8% to 6.4%. However, some patients showed a slight decrease in blood flow. When the data were analysed in terms of regional cerebral blood flow, we found that the most significant increase in blood flow occurred in the frontal lobes (P = 0.02).

Nurturing syndrome: a form of pathological bereavement with delusions in Alzheimer's disease.

We report the history and neuroimaging profile of two patients, CS and KC who developed an unusual bereavement reaction with delusions following the deaths of their husbands. Their neuropsychological profiles met criteria for dementia of Alzheimer type. The delusions about their husbands' existence were persistent despite a considerable passage of time after their deaths. SPECT investigation showed reduction of blood flow in the frontal area in both patients when compared with demented controls and in particular a significant reduction in perfusion was observed in the right frontal regions. We argue that this significant reduction of blood flow in the right frontal area, in the context of severe cognitive deterioration, might be responsible for their delusional reaction to bereavement which we interpret as result of reality monitoring and episodic memory failure.

Use of palatal lift and palatal augmentation prostheses to improve dysarthria in patients with amyotrophic lateral sclerosis: a case series.

STATEMENT OF PROBLEM: Amyotrophic Lateral Sclerosis (ALS) is a progressive, adult onset neurodegenerative disorder manifesting itself as a loss of motor capabilities and untimely death. The dysarthria seen in patients with ALS who have bulbar symptoms causes severe problems with communication. The struggle to be understood comes at a time when progressive cumulative disabilities make communication with family, friends, and healthcare workers vital. The use of palatal lift/augmentation prostheses for dysarthria in ALS is not a frequently requested procedure by neurologists. PURPOSE: The purpose of this retrospective outcomes study was to evaluate the effectiveness of this treatment on improving speech function and intelligibility in this group of patients. This study also reviews the history, incidence, pathogenesis, and speech characteristics of the patient with ALS. METHODS: A retrospective study of 25 patients treated with a prosthesis was performed using chart reviews and phone/office interviews to evaluate the efficacy of a palatal lift and/or augmentation prosthesis to improve speech in ALS patients. RESULTS: Twenty-one patients (84%) treated with a palatal lift demonstrated improvement in their dysarthria, specifically in reduction of hypernasality, with 19 (76%) benefiting at least moderately for 6 months. Of the 10 patients treated with a combination palatal lift and augmentation prosthesis, 6 (60%) demonstrated improvement in articulation. A majority of patients indicated it was easier to speak with less effort involved when wearing the prosthesis. CONCLUSION: On the basis of this preliminary retrospective study, the use of a palatal lift/augmentation prosthesis should be considered in ALS patients with dysarthria.