[Relationship between body condition of dairy cows in the peripartum period and selected metabolic parameters in consideration of different breeds]. / Zusammenhänge zwischen Körperkondition von Milchkühen im peripartalen Zeitraum und ausgewählten Stoffwechselparametern unter Berücksichtigung verschiedener Rassen.

OBJECTIVE: The results of this study describe the relationship between the body condition of dairy cows and selected metabolic parameters during the peri- and post-partum period with special consideration of 3 local dairy cow breed in Upper Bavaria and the Allgau. MATERIAL AND METHODS: Three local dairy cattle breeds (Swiss Brown (BV), Simmental (FL), Holstein Friesian (HF)) were examined on 68 farms in southern Germany for 7 consecutive weeks. In dry cows as well as lactating cows (5.-65. day in milk), following blood parameters were investigated: beta-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), creatinine, aspartate aminotransferase, gamma-glutamyltransferase, glutamate dehydrogenase, total protein, albumin, creatine kinase. In addition, body condition (body condition score [BCS] and back fat thickness [BFT]) were recorded. Exploratory and descriptive statistics were used for data analysis. RESULTS: Concerning the difference in condition before and after calving, the FL showed the smallest difference in RFD. For FL and BV a trend towards higher BFT values could be seen in first lactating cows. For FL and HF, the NEFA values of the later lactating cows were below those of the first lactating cows. The higher lactating cows of BV and FL had higher BHB values. The correlation between BFT and BCS showed the highest R2 (0.53) in the HF cows. BV and FL were below at 0.42 and 0.37. BCS and BFT could not be predicted by the variables NEFA, BHB and liver enzymes. BHB levels of all 3 breeds increased at weeks 2-4 post-partum. The NEFA values for all 3 breeds increased primarily in the 1st-3rd week p.p. in parallel to when the BFT p.p. decreased. NEFA values were highest when body condition declined and therefore when fat mobilization peaked. In BV and HF, there was a constant increase in GLDH when the p.p. BCS difference was there. CONCLUSION AND CLINICAL RELEVANCE: Body condition assessment (BCS at herd and animals` level, BFT at animal level) is an important tool for animal health monitoring. Due to the recognizable breed specificity, the dairy herds can be dealt with more explicitly. The aim is to optimally influence the energy balance of the cow during early lactation in order maintain the health of the animal and its organ systems.

In Silico CRISPR-Cas-Mediated Base Editing Strategies for Early-Onset, Severe Cone-Rod Retinal Degeneration in Three Crumbs homolog 1 Patients, including the Novel Variant c.2833G>A.

Pathogenic variants in the Crumbs homolog 1 (CRB1) gene lead to severe, childhood-onset retinal degeneration leading to blindness in early adulthood. There are no approved therapies, and traditional adeno-associated viral vector-based gene therapy approaches are challenged by the existence of multiple CRB1 isoforms. Here, we describe three CRB1 variants, including a novel, previously unreported variant that led to retinal degeneration. We offer a CRISPR-Cas-mediated DNA base editing strategy as a potential future therapeutic approach. This study is a retrospective case series. Clinical and genetic assessments were performed, including deep phenotyping by retinal imaging. In silico analyses were used to predict the pathogenicity of the novel variant and to determine whether the variants are amenable to DNA base editing strategies. Case 1 was a 24-year-old male with cone-rod dystrophy and retinal thickening typical of CRB1 retinopathy. He had a relatively preserved central outer retinal structure and a best corrected visual acuity (BCVA) of 60 ETDRS letters in both eyes. Genetic testing revealed compound heterozygous variants in exon 9: c.2843G>A, p.(Cys948Tyr) and a novel variant, c.2833G>A, p.(Gly945Arg), which was predicted to likely be pathogenic by an in silico analysis. Cases 2 and 3 were two brothers, aged 20 and 24, who presented with severe cone-rod dystrophy and a significant disruption of the outer nuclear layers. The BCVA was reduced to hand movements in both eyes in Case 2 and to 42 ETDRS letters in both eyes in Case 3. Case 2 was also affected with marked cystoid macular lesions, which are common in CRB1 retinopathy, but responded well to treatment with oral acetazolamide. Genetic testing revealed two c.2234C>T, p.(Thr745Met) variants in both brothers. As G-to-A and C-to-T variants, all three variants are amenable to adenine base editors (ABEs) targeting the forward strand in the Case 1 variants and the reverse strand in Cases 2 and 3. Available PAM sites were detected for KKH-nSaCas9-ABE8e for the c.2843G>A variant, nSaCas9-ABE8e and KKH-nSaCas9-ABE8e for the c.2833G>A variant, and nSpCas9-ABE8e for the c.2234C>T variant. In this case series, we report three pathogenic CRB1 variants, including a novel c.2833G>A variant associated with early-onset cone-rod dystrophy. We highlight the severity and rapid progression of the disease and offer ABEs as a potential future therapeutic approach for this devastating blinding condition.

[Care-related Termination of Employment among Informal Caregivers of Elderly Persons: Identification of a Risk Profile]. / Pflegebedingte Beendigung der Erwerbstätigkeit pflegender Angehöriger: Identifikation eines Risikoprofils.

OBJECTIVE: Employed informal caregivers often experience role conflicts between caring for an elderly person in need of care at home and their employment. The goal of this paper was to identify a risk profile of care-related termination of employment. METHODS: Analyses are based on the cross-sectional Benefits of Being a Caregiver Study (October 2019 - March 2020) with data from 481 informal caregivers of elderly persons in need of care. The data collected relate to characteristics of the care recipient, the informal caregiver, and the caregiving situation, as well as aspects of the employment situation. The risk profile of care-related cessation of employment is based on a binary logistic regression. RESULTS: Approximately one in nine in the present sample (n=55) terminated employment because of having to offer informal care to an elderly person at home. Factors characterizing the risk profile of a care-related termination of employment were female gender of the caregivers, younger age of the care receiver, co-residence with the care receiver, and a higher care level of the care receiver. CONCLUSIONS: In order to reduce care-related cessation of employment, support and relief services need to be adapted to the factors of the identified risk profile. In particular, the form and content of informal caregiver counselling should be modified in order to reach informal caregivers at an early stage. Adapted support programs should focus on and reach in particular female employed caregivers.

[Reconciling Employment and Informal Caregiving for an Elderly Person at Home: Factors Influencing a Reduction in Employment due to Caregiving and Work-Related Wishes of Informal Caregivers]. / Vereinbarkeit von Erwerbstätigkeit und häuslicher Pflege: Einflussfaktoren auf eine pflegebedingte Erwerbstätigkeitsreduktion und arbeitsbezogene Wünsche pflegender Angehöriger.

OBJECTIVE: Reconciling informal caregiving and gainful employment is a challenge for many informal caregivers. The goals of this paper are to identify factors influencing care-related employment reduction, and to record work-related wishes for improving the compatibility of informal caregiving and being employed. METHODS: Analyses were based on the cross-sectional Benefits of Being a Caregiver Study of 426 employed caregivers of an older person in need of care. Data were collected on characteristics of the care receivers and caregivers, and aspects of the caregiving and employment situation. Potential influencing factors of care-related employment reduction (n=426) were analyzed using binary logistic regression. The wishes regarding the compatibility of informal care at home and employment were examined descriptively using structured content analysis according to Mayring. RESULTS: One quarter of the employed informal caregivers (n=108) reduced their hours of employment due to the demands of caregiving. The profile of influencing factors of a care-related employment reduction was composed of a higher number of working hours, higher effort for activities of daily living, and co-residence with the care receiver. Employed caregivers primarily expressed a desire for flexibility in working hours, a reduction in working hours, and some concessions with regard to absenteeism. CONCLUSIONS: Relieving the burden on caregivers in the activities of daily living in form of formal and informal support services can probably reduce the likelihood of a care-related reduction in gainful employment.

[Sometimes, I Also Drink Water - Alcohol Consumption of People With Mild Cognitive Impairment]. / Manchmal trink ich auch Wasser ­ Alkoholkonsum von Menschen mit leichter kognitiver Beeinträchtigung.

AIM: Risky alcohol consumption increases the risk of dementia for people with mild cognitive impairment (MCI). The aim of this study is to assess alcohol consumption in people with MCI. METHODS: Socio-demographics, 12-month prevalence, 30-d prevalence, prevalence of risky consumption (>10 g/20 g/d pure alcohol for women/men) and binge drinking (≥50 g pure alcohol on one occasion) were recorded in 270 people (≥60 years) with MCI from the German RCT "Brainfit-Nutrition" in 2022. RESULTS: Approximately half of the people with MCI (50.8%) drink at least once a week. About one fifth (17.0%) of participants met the criterion for binge drinking; every third woman (34.8%) and every fifth man (18.6%) crossed the line to risky consumption in the last 30 d. DISCUSSION: Generally, people with MCI show similar consumption prevalence as the 65+German general population. However, the prevalence of risky consumption in women with MCI is significantly higher.

Optical Coherence Tomography Feature of Retinoschisis in CRB1-Associated Maculopathy.

This case report describes 2 individuals with hyperreflective columns in the outer nuclear layer observed on optical coherence tomography and possible implications for CRB1-associated maculopathy.

Are Protective Activities and Limitations in Practical Skills of Daily Living Associated with the Cognitive Performance of People with Mild Cognitive Impairment? Baseline Results from the BrainFit-Nutrition Study.

Limitations in daily living have not yet been described adequately for mild cognitive impairment (MCI). In this study, we investigated first, time spent on protective activities (social, mental, and physical) and second, limitations in practical skills of daily living, both for people with MCI. We used baseline data from 270 individuals who participated in the randomized controlled trial BrainFit-Nutrition. The Montreal Cognitive Assessment (MoCA) was used to identify people with MCI. Participants were asked how much time they spent engaged in social, mental, and physical activities each week. Furthermore, the Bayer-ADL scale was used to quantify deficits in activities of daily living (ADLs). Regarding protection, the number of hours spent engaged in the three activity areas was significantly correlated with the cognitive performance in people with MCI. Social activities were positively associated with current cognitive performance. Concerning the limitations in practical skills of daily living, older and more cognitively impaired individuals were affected. Memory and orientation appear to be among the first practical skills of daily living that become impaired in people with MCI. Treatment recommendations for people with MCI include an increase in social, mental, and physical activities as well as the promotion of a healthy lifestyle.

Programmable RNA editing with endogenous ADAR enzymes - a feasible option for the treatment of inherited retinal disease?

RNA editing holds great promise for the therapeutic correction of pathogenic, single nucleotide variants (SNV) in the human transcriptome since it does not risk creating permanent off-targets edits in the genome and has the potential for innovative delivery options. Adenine deaminases acting on RNA (ADAR) enzymes catalyse the most widespread form of posttranscriptional RNA editing in humans and their ability to hydrolytically deaminate adenosine to inosine in double stranded RNA (dsRNA) has been harnessed to change pathogenic single nucleotide variants (SNVs) in the human genome on a transcriptional level. Until now, the most promising target editing rates have been achieved by exogenous delivery of the catalytically active ADAR deaminase domain (ADARDD) fused to an RNA binding protein. While it has been shown that endogenous ADARs can be recruited to a defined target site with the sole help of an ADAR-recruiting guide RNA, thus freeing up packaging space, decreasing the chance of an immune response against a foreign protein, and decreasing transcriptome-wide off-target effects, this approach has been limited by a low editing efficiency. Through the recent development of novel circular ADAR-recruiting guide RNAs as well as the optimisation of ADAR-recruiting antisense oligonucleotides, RNA editing with endogenous ADAR is now showing promising target editing efficiency in vitro and in vivo. A target editing efficiency comparable to RNA editing with exogenous ADAR was shown both in wild-type and disease mouse models as well as in wild-type non-human primates (NHP) immediately following and up to 6 weeks after application. With these encouraging results, RNA editing with endogenous ADAR has the potential to present an attractive option for the treatment of inherited retinal diseases (IRDs), a field where gene replacement therapy has been established as safe and efficacious, but where an unmet need still exists for genes that exceed the packaging capacity of an adeno associated virus (AAV) or are expressed in more than one retinal isoform. This review aims to give an overview of the recent developments in the field of RNA editing with endogenous ADAR and assess its applicability for the field of treatment of IRD.

Predictors of expressed, felt, and normative needs for informal caregiver counseling : Domestic care for people aged 65+ years.

BACKGROUND: Informal caregivers (CGs) often fail to recognize or express a need for informal caregiver counseling (ICC) but ICC is an essential but relatively rarely used support service for CGs. OBJECTIVE: Our aim is to identify predictors of CGs' need for ICC. Stirling et al.'s need model, which includes three needs (expressed, felt, and normative), serves as a theoretical basis. MATERIAL AND METHODS: Analyses are based on cross-sectional data (n = 958) from the "Benefits of being a caregiver" study. Predictors of the need to use ICC were analyzed with binary logistic regression. A sensitivity analysis using multiple linear regression was performed for the metric value of normative needs. RESULTS: We found that 6.8% of CGs currently or have recently used ICC. This expressed need was related to higher education and higher effort in instrumental activities; 24.1% of CGs reported an intention to use ICC in the future. This felt need was related to male gender, lower care level, more problem-focused coping, and a desire for more informal help. Objective need for ICC (normative need), which was related to a higher burden of care, less experienced benefits, and negative relationship quality, was reported by 21.4% of CGs. According to a sensitivity analysis, higher education, a desire for informal help, and living in separate households also predicted a normative need for counseling. DISCUSSION: Current utilization is significantly lower than the subjectively perceived and objectively existing need for ICC. The identified predictors provide initial strategies for motivating more CGs to use ICC.

Computerised cognitive training tools and online nutritional group counselling for people with mild cognitive impairment: study protocol of a completely digital, randomised, controlled trial.

INTRODUCTION: People with mild cognitive impairment (MCI) are at increased risk of decreasing cognitive functioning. Computerised cognitive training (CCT) and nutrition have been shown to improve the cognitive capacities of people with MCI. For each variable, we developed two kinds of interventions specialised for people with MCI (CCT: 'individualised' CCT; nutrition: a whole-food, plant-based diet). Additionally, there are two kinds of active control measures (CCT: 'basic' CCT; nutrition: a healthy diet following the current guidelines of the German Nutrition Society). The aim of this study is to investigate the effects of the two interventions on cognition in people with MCI in a 2×2 randomised controlled trial with German participants. METHODS AND ANALYSIS: Participants will be community-dwelling individuals with a psychometric diagnosis of MCI based on the Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination. With N=200, effects with an effect size of f≥0.24 (comparable to Cohen's d≥0.48) can be detected. Screening, baseline, t6 and t12 testing will be conducted via a videoconferencing assessment, telephone, and online survey. Participants will be randomly allocated to one of four groups and will receive a combination of CCT and online nutritional counselling. The CCT can be carried out independently at home on a computer, laptop, or tablet. Nutrition counselling includes 12 online group sessions every fortnight for 1.5 hours. The treatment phase is 6 months with follow-ups after six and 12 months after baseline. ETHICS AND DISSEMINATION: All procedures were approved by the Friedrich-Alexander-Universität Erlangen-Nürnberg Ethics Committee (Ref. 21-318-1-B). Written informed consent will be obtained from all participants. Results will be published in peer-reviewed scientific journals, conference presentations. TRIAL REGISTRATION NUMBER: ISRCTN10560738.

In Silico Analysis of Pathogenic CRB1 Single Nucleotide Variants and Their Amenability to Base Editing as a Potential Lead for Therapeutic Intervention.

Mutations in the Crumbs homolog 1 (CRB1) gene cause both autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Since three separate CRB1 isoforms are expressed at meaningful levels in the human retina, base editing shows promise as a therapeutic approach. This retrospective analysis aims to summarise the reported pathogenic CRB1 variants and investigate their amenability to treatment with currently available DNA base editors. Pathogenic single nucleotide variants (SNVs) were extracted from the Leiden open-source variation database (LOVD) and ClinVar database and coded by mutational consequence. They were then analyzed for their amenability to currently available DNA base editors and available PAM sites from a selection of different Cas proteins. Of a total of 1115 unique CRB1 variants, 69% were classified as pathogenic SNVs. Of these, 62% were amenable to currently available DNA BEs. Adenine base editors (ABEs) alone have the potential of targeting 34% of pathogenic SNVs; 19% were amenable to a CBE while GBEs could target an additional 9%. Of the pathogenic SNVs targetable with a DNA BE, 87% had a PAM site for a Cas protein. Of the 33 most frequently reported pathogenic SNVs, 70% were targetable with a base editor. The most common pathogenic variant was c.2843G>A, p.Cys948Arg, which is targetable with an ABE. Since 62% of pathogenic CRB1 SNVs are amenable to correction with a base editor and 87% of these mutations had a suitable PAM site, gene editing represents a promising therapeutic avenue for CRB1-associated retinal degenerations.

Laparoscopic liver resection in Caroli disease: A single-centre case series.

BACKGROUND: Liver resection is the treatment of choice for patients with localised Caroli disease. While liver resection was traditionally performed as open procedure, this case series aims to evaluate the safety and efficacy of minimally invasive, laparoscopic liver surgery in these patients. METHODS: A systematic review of electronic case files of patients seen between April 2015 and December 2017 at the Department of Surgery, Charité University Hospital Berlin, was conducted. Patients with Caroli disease in whom laparoscopic liver resection had been performed were identified and analysed in this single-centre case series. RESULTS: Seven patients who underwent laparoscopic liver surgery for Caroli syndrome were identified and presented with a median age of 49 (range = 44-66) years, of which four (57%) were female. Preoperatively, six patients were classified as the American Society of Anaesthesiologists (ASA) 2 and one patient as ASA 3. Two operations were performed as single-incision laparoscopic surgery, whereas the others were done as multi-incision laparoscopic surgery. One patient required a conversion to an open procedure. The length of operation varied between patients, ranging from 128 to 758 min (median = 355). The length of stay in the intensive care unit ranged from 0 to 2 days. Two patients presented with post-operative complications (Clavien-Dindo Grade ≥3a), whereas no patient died. In histopathological analysis, all patients demonstrated characteristic findings of Caroli disease and no cholangiocarcinoma was found. CONCLUSION: These results indicate that minimally invasive, laparoscopic liver surgery is a safe and efficacious treatment option for patients with Caroli disease who require liver resection.

Reduced spontaneous perspective taking in schizophrenia.

Judgments about another person's visual perspective are impaired when the self-perspective is inconsistent with the other-perspective. This is a robust finding in healthy samples as well as in schizophrenia (SZ). Studies show evidence for the existence of a reverse effect, where an inconsistent other-perspective impairs the self-perspective. Such spontaneous perspective taking processes are not yet explored in SZ. In the current fMRI experiment, 24 healthy and 24 schizophrenic participants performed a visual perspective taking task in the scanner. Either a social or a non-social stimulus was presented and their visual perspectives were consistent or inconsistent with the self-perspective of the participant. We replicated previous findings showing that healthy participants show increased reaction times when the human avatar's perspective is inconsistent to the self-perspective. Patients with SZ, however, did not show this effect, neither in the social nor in the non-social condition. BOLD responses revealed similar patterns in occipital areas and group differences were identified in the middle occipital gyrus. These findings suggest that patients with SZ are less likely to spontaneously compute the visual perspectives of others.

Minimally Invasive Liver Surgery in Elderly Patients-A Single-Center Experience.

BACKGROUND: In recent years, laparoscopic liver resection has elicited growing attention as a safe procedure for various forms of hepatic resection. In the context of an aging population, this study aims to evaluate outcomes in elderly patients (>70 y) compared with younger patients (≤70 y). METHODS: All consecutive patients undergoing minimally invasive liver resections between December 2013 and January 2018 at the Department of Surgery, Charité-Universitätsmedizin Berlin, were included in this analysis. Patients' characteristics, such as body mass index, American Society of Anesthesiologists classification, as well as underlying liver disease and function, were examined and the perioperative outcomes of patients aged >70 y (group 1; G1) contrasted with patients aged ≤ 70 y (group 2; G2). RESULTS: Of 250 patients, 67 were >70 y old (G1) and 183 were ≤70 y old (G2). Patients in G1 were characterized by a higher body mass index (27.6 kg/m2versus 24.9 kg/m2; P = 0.004) and impaired physical states (American Society of Anesthesiologists score III/IV; 60% versus 37%; P = 0.002) when compared with group 2. G1 also exhibited higher rates of primary and secondary hepatic malignancies (G1: n = 62; 92.5%; G2: n = 115, 62.8%; P = 0.031) in addition to higher rates of cirrhosis (G1: n = 30, 44.8%; G2: n = 38, 20.8%; P = <0.001). The rate of major complications (Dindo-Clavien grade ≥ III) was similar between both groups (P = 0.58), with no differences regarding resection extent (P = 0.469). No difference was evident with regard to the median intensive care unit (median 1 versus 1 d; range, G1, 0-8 d, G2, 0-23 d; P = 0.1). However, we observed a significant longer hospital stay in G1 of 1 d (median 8 versus 9 d; G1 range: 4-35 d: G2 range: 4-59 d; P = 0.015). CONCLUSIONS: Minimally invasive liver resection is a feasible and safe procedure in elderly patients despite this age group exhibiting a higher rate of primary and secondary malignancy and cirrhosis, as well as an overall more severely compromised physical health when compared with patients under the age of 70 y. Therefore, it stands to reason that patients in poorer general health might particularly benefit from a minimally invasive approach.

Regional brain volumetry and brain function in severely brain-injured patients.

OBJECTIVE: The relationship between residual brain tissue in patients with disorders of consciousness (DOC) and the clinical condition is unclear. This observational study aimed to quantify gray (GM) and white matter (WM) atrophy in states of (altered) consciousness. METHODS: Structural T1-weighted magnetic resonance images were processed for 102 severely brain-injured and 52 healthy subjects. Regional brain volume was quantified for 158 (sub)cortical regions using Freesurfer. The relationship between regional brain volume and clinical characteristics of patients with DOC and conscious brain-injured patients was assessed using a linear mixed-effects model. Classification of patients with unresponsive wakefulness syndrome (UWS) and minimally conscious state (MCS) using regional volumetric information was performed and compared to classification using cerebral glucose uptake from fluorodeoxyglucose positron emission tomography. For validation, the T1-based classifier was tested on independent datasets. RESULTS: Patients were characterized by smaller regional brain volumes than healthy subjects. Atrophy occurred faster in UWS compared to MCS (GM) and conscious (GM and WM) patients. Classification was successful (misclassification with leave-one-out cross-validation between 2% and 13%) and generalized to the independent data set with an area under the receiver operator curve of 79% (95% confidence interval [CI; 67-91.5]) for GM and 70% (95% CI [55.6-85.4]) for WM. INTERPRETATION: Brain volumetry at the single-subject level reveals that regions in the default mode network and subcortical gray matter regions, as well as white matter regions involved in long range connectivity, are most important to distinguish levels of consciousness. Our findings suggest that changes of brain structure provide information in addition to the assessment of functional neuroimaging and thus should be evaluated as well. Ann Neurol 2018;83:842-853.

High Symmetry of Visual Acuity and Visual Fields in RPGR-Linked Retinitis Pigmentosa.

Purpose: Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause 70% to 90% of X-linked retinitis pigmentosa (XLRP3) cases, making this gene a high-yield target for gene therapy. This study analyzed the utility of relevant clinical biomarkers to assess symmetry and rate of progression in XLRP3. Methods: A retrospective, cross-sectional analysis of 50 XLRP3 patients extracted clinical data including visual acuity (VA), visual fields (I4e and III4e targets), foveal thickness, and ERG data points alongside molecular genetic data. Symmetry was assessed by using linear regression analysis. Kaplan-Meier survival curves (KMCs) and generalized linear mixed model calculations were used to describe disease progression. Results: Ninety-six percent of patients exhibited a rod-cone phenotype, and 4% a cone-rod phenotype. Open reading frame 15 (ORF15) was confirmed as a mutational hotspot within RPGR harboring 73% of exonic mutations. Significant variability, but no clear genotype-phenotype relationship, could be shown between mutations located in exons 1-14 versus ORF15. All biomarkers suggested a high degree of symmetry between eyes but demonstrated different estimates of disease progression. VA and foveal thickness, followed by perimetry III4e, were the most useful endpoints to evaluate progression. KMC estimates predicted a loss of 6/6 vision at a mean of 34 years (±2.9; 95% confidence interval). Conclusions: XLRP3 affects retinal structure and function symmetrically, supporting the use of the fellow eye as an internal control in interventional trials. VA and kinetic visual fields (III4e) seem promising functional outcome measures to assess disease progression. KMC analysis predicted the most severe decline in vision between the third and fourth decade of life.

Codon-Optimized RPGR Improves Stability and Efficacy of AAV8 Gene Therapy in Two Mouse Models of X-Linked Retinitis Pigmentosa.

X-linked retinitis pigmentosa (XLRP) is generally a severe form of retinitis pigmentosa, a neurodegenerative, blinding disorder of the retina. 70% of XLRP cases are due to mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGRORF15). Despite successful RPGRORF15 gene replacement with adeno-associated viral (AAV) vectors being established in a number of animal models of XLRP, progression to human trials has not yet been possible. The inherent sequence instability in the purine-rich region of RPGRORF15 (which contains highly repetitive nucleotide sequences) leads to unpredictable recombination errors during viral vector cloning. While deleted RPGR may show some efficacy in animal models, which have milder disease, the therapeutic effect of a mutated RPGR variant in patients with XLRP cannot be predicted. Here, we describe an optimized gene replacement therapy for human XLRP disease using an AAV8 vector that reliably and consistently produces the full-length correct RPGR protein. The glutamylation pattern in the RPGR protein derived from the codon-optimized sequence is indistinguishable from the wild-type variant, implying that codon optimization does not significantly alter post-translational modification. The codon-optimized sequence has superior stability and expression levels in vitro. Significantly, when delivered by AAV8 vector and driven by the rhodopsin kinase promoter, the codon-optimized RPGR rescues the disease phenotype in two relevant animal models (Rpgr-/y and C57BL/6JRd9/Boc) and shows good safety in C57BL6/J wild-type mice. This work provides the basis for clinical trial development to treat patients with XLRP caused by RPGR mutations.

Application of whole-exome sequencing to unravel the molecular basis of undiagnosed syndromic congenital neutropenia with intellectual disability.

Neutropenia can be qualified as congenital when of neonatal onset or when associated with extra-hematopoietic manifestations. Overall, 30% of patients with congenital neutropenia (CN) remain without a molecular diagnosis after a multidisciplinary consultation and tedious diagnostic strategy. In the rare situations when neutropenia is identified and associated with intellectual disability (ID), there are few diagnostic hypotheses to test. This retrospective multicenter study reports on a clinically heterogeneous cohort of 10 unrelated patients with CN associated with ID and no molecular diagnosis prior to whole-exome sequencing (WES). WES provided a diagnostic yield of 40% (4/10). The results suggested that in many cases neutropenia and syndromic manifestations could not be assigned to the same molecular alteration. Three sub-groups of patients were highlighted: (i) severe, symptomatic chronic neutropenia, detected early in life, and related to a known mutation in the CN spectrum (ELANE); (ii) mild to moderate benign intermittent neutropenia, detected later, and associated with mutations in genes implicated in neurodevelopmental disorders (CHD2, HUWE1); and (iii) moderate to severe intermittent neutropenia as a probably undiagnosed feature of a newly reported syndrome (KAT6A). Unlike KAT6A, which seems to be associated with a syndromic form of CN, the other reported mutations may not explain the entire clinical picture. Although targeted gene sequencing can be discussed for the primary diagnosis of severe CN, we suggest that performing WES for the diagnosis of disorders associating CN with ID will not only provide the etiological diagnosis but will also pave the way towards personalized care and follow-up. © 2016 Wiley Periodicals, Inc.

Testing Proposed Neuronal Models of Effective Connectivity Within the Cortico-basal Ganglia-thalamo-cortical Loop During Loss of Consciousness.

In recent years, a number of brain regions and connectivity patterns have been proposed to be crucial for loss and recovery of consciousness but have not been compared in detail. In a 3 T resting-state functional magnetic resonance imaging paradigm, we test the plausibility of these different neuronal models derived from theoretical and empirical knowledge. Specifically, we assess the fit of each model to the dynamic change in effective connectivity between specific cortical and subcortical regions at different consecutive levels of propofol-induced sedation by employing spectral dynamic causal modeling. Surprisingly, our findings indicate that proposed models of impaired consciousness do not fit the observed patterns of effective connectivity. Rather, the data show that loss of consciousness, at least in the context of propofol-induced sedation, is marked by a breakdown of corticopetal projections from the globus pallidus. Effective connectivity between the globus pallidus and the ventral posterior cingulate cortex, present during wakefulness, fades in the transition from lightly sedated to full loss of consciousness and returns gradually as consciousness recovers, thereby, demonstrating the dynamic shift in brain architecture of the posterior cingulate "hub" during changing states of consciousness. These findings highlight the functional role of a previously underappreciated direct pallido-cortical connectivity in supporting consciousness.