Rett syndrome diagnostic odyssey: Limitations of NextGen sequencing.

Typical (or classic) Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by a period of regression, partial or complete loss of purposeful hand movements, and acquired speech, impaired gait, and stereotyped hand movements. In over 95% of typical RTT, a pathogenic variant is found in the methyl-CPG binding protein 2 gene (MECP2). Here, we describe a young woman with clinically diagnosed typical RTT syndrome who lacked a genetic diagnosis despite 20 years of investigation and multiple rounds of sequencing the MECP2 gene. Recently, additional genetic testing using next-generation sequencing was completed, which revealed a partial insertion of the BCL11A gene within exon 4 of MECP2, resulting in a small deletion in MECP2, causing likely disruption of MeCP2 function due to a frameshift. This case demonstrates the ever-changing limitations of genetic testing, as well as the importance of continual pursuit of a diagnosis as technologies improve and are more widely utilized.

Secondary use of genomic data: patients' decisions at point of testing and perspectives to inform international data sharing.

International sharing of genomic data files arising from clinical testing of patients is essential to further improve genomic medicine. Whilst the general public are reluctant to donate DNA for research, the choices patients actually make about sharing their clinical genomic data for future re-use (research or clinical) are unknown. We ascertained the data-sharing choices of 1515 patients having genomic testing for inherited conditions or cancer treatment from clinical consent forms. To understand the experiences and preferences of these patients, surveys were administered after test consent (RR 73%). Almost all patients (98%) consented to share their data. Survey respondents' decision recall was high (90%), but poorer if English was an additional language (p < 0.001). Parents deciding on behalf of children were over-represented amongst data-sharing decliners (p = 0.047) and decliners were more likely to believe that stored data could be easily reidentified (p < 0.001). A quarter of respondents did not know if reidentification would be easy and 44% of them were concerned about this possibility. Of those willing to share data overseas (60%), 23% indicated the recipient researcher's country would affect their decision. Most respondents (89%) desired some ongoing control over research use of their data. Four preliminary data-sharing profiles emerged; their further development could inform tailored patient resources. Our results highlight considerations for establishment of systems to make clinical genomic data files available for reanalysis locally and across borders. Patients' willingness to share their data - and value of the resulting research - should encourage clinical laboratories to consider sharing data systematically for secondary uses.

Adult-onset Krabbe disease presenting as isolated sensorimotor demyelinating polyneuropathy: A case report.

Krabbe disease is a rare autosomal recessive neurodegenerative disease, caused by mutations in the GALC gene, which encodes for the lysosomal enzyme galactocerebrosidase. Typical clinical manifestations of Krabbe include psychomotor deterioration, visual loss, seizures, and spasticity, that result from central nervous system demyelination. We report a case of a 35-year-old male with Krabbe who presented in adulthood with isolated severe, upper extremity predominant demyelinating sensorimotor polyneuropathy and did not develop other distinguishing clinical or radiological features of Krabbe until the later stages of the disease. The patient's diagnostic odyssey lasted 13 years from presentation to diagnosis, which was ultimately determined with the use of whole exome sequencing (WES) at the age of 48 years. The expanding phenotypic spectrum of adult-onset Krabbe Disease (AOKD) presents a diagnostic challenge that can lead to diagnostic delays and potentially affect treatment options. Our patient's case underscores the importance of pursuing WES in those with undiagnosed progressive neuromuscular disorders.

A predictive model and a field study on heterogeneous slug distribution in arable fields arising from density dependent movement.

Factors and processes determining heterogeneous ('patchy') population distributions in natural environments have long been a major focus in ecology. Existing theoretical approaches proved to be successful in explaining vegetation patterns. In the case of animal populations, existing theories are at most conceptual: they may suggest a qualitative explanation but largely fail to explain patchiness quantitatively. We aim to bridge this knowledge gap. We present a new mechanism of self-organized formation of a patchy spatial population distribution. A factor that was under-appreciated by pattern formation theories is animal sociability, which may result in density dependent movement behaviour. Our approach was inspired by a recent project on movement and distribution of slugs in arable fields. The project discovered a strongly heterogeneous slug distribution and a specific density dependent individual movement. In this paper, we bring these two findings together. We develop a model of density dependent animal movement to account for the switch in the movement behaviour when the local population density exceeds a certain threshold. The model is fully parameterized using the field data. We then show that the model produces spatial patterns with properties closely resembling those observed in the field, in particular to exhibit similar values of the aggregation index.

Modifiable Comorbidities Associated with Cognitive Decline in Parkinson's Disease.

BACKGROUND: Cognitive impairment (CI) is one of the most feared and debilitating complications of PD. No therapy has been shown to slow or prevent CI in PD. OBJECTIVE: To determine associations between modifiable comorbidities, including cardiovascular disease risk factors, mood disorders, and sleep characteristics, and rate of cognitive decline in Parkinson's disease (PD). METHODS: Data from the Parkinson's Progression Markers Initiative (PPMI) cohort was queried for baseline cardiovascular disease risk factors, mood disorders, and sleep characteristics. Linear mixed- effects models (LME) were used to examine the association between baseline factors and change in cognition, evaluated by the Montreal Cognitive Assessment (MoCA) over time. Baseline comorbidities found to affect MoCA decline were assessed for an association with focal cognitive domains using LME. RESULTS: Higher Body Mass Index (BMI) (ß = -0.009, P = 0.039), State Trait Anxiety Inventory (STAI) (ß = -0.005, P < 0.001), Geriatric Depression Scale (GDS) (ß = -0.034, P < 0.001), Epworth Sleepiness Scale (ESS) (ß = -0.017, P = 0.003), and REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) (ß = -0.037, P < 0.001) were associated with faster rates of MoCA decline. Using established cut-offs for clinically significant symptoms, being overweight, or the presence of depression, excessive day time sleepiness (EDS), and possible REM sleep behavior disorder (pRBD), were all associated with faster rate of cognitive decline. CONCLUSION: Several modifiable baseline comorbidities are associated with faster rate of CI over time in patients with PD. These associations identify potential opportunities for early intervention that could influence CI in PD.

Stability of Patches of Higher Population Density within the Heterogenous Distribution of the Gray Field Slug Deroceras reticulatum in Arable Fields in the UK.

Exploitation of heterogenous distributions of Deroceras reticulatum, in arable fields by targeting molluscicide applications toward areas with higher slug densities, relies on these patches displaying sufficient spatio-temporal stability. Regular sampling of slug activity/distribution was undertaken using 1 ha rectangular grids of 100 refuge traps established in 22 commercial arable field crops. Activity varied significantly between the three years of the study, and the degree of aggregation (Taylor's Power Law) was higher in fields with higher mean trap catches. Hot spot analysis detected statistically significant spatial clusters in all fields, and in 162 of the 167 individual assessment visits. The five assessment visits in which no clusters were detected coincided with low slug activity (≤0.07 per trap). Generalized Linear Models showed significant spatial stability of patches in 11 fields, with non-significant fields also characterized by low slug activity (≤1.2 per trap). Mantel's permutation tests revealed a high degree of correlation between location of individual patches between sampling dates. It was concluded that patches of higher slug density were spatio-temporally stable, but detection using surface refuge traps (which rely on slug activity on the soil surface) was less reliable when adverse environmental conditions resulted in slugs retreating into the upper soil horizons.

Movement patterns of the grey field slug (Deroceras reticulatum) in an arable field.

We report the results of an experiment on radio-tracking of individual grey field slugs in an arable field and associated data modelling designed to investigate the effect of slug population density in their movement. Slugs were collected in a commercial winter wheat field in which a 5x6 trapping grid had been established with 2m distance between traps. The slugs were taken to the laboratory, radio-tagged using a recently developed procedure, and following a recovery period released into the same field. Seventeen tagged slugs were released singly (sparse release) on the same grid node on which they had been caught. Eleven tagged slugs were released as a group (dense release). Each of the slugs was radio-tracked for approximately 10 h during which their position was recorded ten times. The tracking data were analysed using the Correlated Random Walk framework. The analysis revealed that all components of slug movement (mean speed, turning angles and movement/resting times) were significantly different between the two treatments. On average, the slugs released as a group disperse more slowly than slugs released individually and their turning angle has a clear anticlockwise bias. The results clearly suggest that population density is a factor regulating slug movement.

Locomotor behaviour promotes stability of the patchy distribution of slugs in arable fields: Tracking the movement of individual Deroceras reticulatum.

BACKGROUND: The distribution of the grey field slug (Deroceras reticulatum Müller) in arable fields is characterised by patches containing higher slug densities dispersed within areas of lower densities. Behavioural responses that lead to the spatial/temporal stability of these patches are poorly understood, thus this study investigated behavioural mechanisms underpinning slug distribution using a new method for long-term tracking of individual slug movement in the field. RESULTS: A technique for implanting radio frequency identification (RFID) tags (each with a unique identification code) beneath the body wall of slugs was developed. Laboratory tests indicated no consistent detrimental effect on survival, feeding, egg laying or locomotor behaviour (velocity, distance travelled). Movement of individual slugs above and below the soil surface was recorded for >5 weeks (in spring and autumn) in winter wheat fields. Most (~80%) foraged within a limited area; and at the end of the observation period were located at a mean distance of 78.7 ± 33.7 cm (spring) or 101.9 ± 24.1 cm (autumn) from their release point. The maximum detected distance from the release point was 408.8 cm. The remaining slugs (~20%) moved further away and ultimately were lost. CONCLUSIONS: RFID tagging allowed continuous tracking of individual slugs, even below the soil surface. Localised movement of 80% of tracked slugs over 5 weeks offers a mechanism promoting stable slug patches in arable crops. Rapid dispersal of the remaining slugs facilitates exchange of individuals between patches. Precision targeting of pesticides at such stable slug patches may facilitate reduced usage. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Towards the Development of a More Accurate Monitoring Procedure for Invertebrate Populations, in the Presence of an Unknown Spatial Pattern of Population Distribution in the Field.

Studies addressing many ecological problems require accurate evaluation of the total population size. In this paper, we revisit a sampling procedure used for the evaluation of the abundance of an invertebrate population from assessment data collected on a spatial grid of sampling locations. We first discuss how insufficient information about the spatial population density obtained on a coarse sampling grid may affect the accuracy of an evaluation of total population size. Such information deficit in field data can arise because of inadequate spatial resolution of the population distribution (spatially variable population density) when coarse grids are used, which is especially true when a strongly heterogeneous spatial population density is sampled. We then argue that the average trap count (the quantity routinely used to quantify abundance), if obtained from a sampling grid that is too coarse, is a random variable because of the uncertainty in sampling spatial data. Finally, we show that a probabilistic approach similar to bootstrapping techniques can be an efficient tool to quantify the uncertainty in the evaluation procedure in the presence of a spatial pattern reflecting a patchy distribution of invertebrates within the sampling grid.

Peripheral blood mononuclear cells derived from grand multigravidae display a distinct cytokine profile in response to P. falciparum infected erythrocytes.

Immunopathology of placental malaria is most significant in women in their first pregnancy especially in endemic areas, due to a lack of protective immunity to Plasmodium falciparum, which is acquired in successive pregnancies. In some studies (but not all), grand multigravidae (defined as 5 or more pregnancies, G5-7) are more susceptible to poor birth outcomes associated with malaria compared to earlier gravidities. By comparing peripheral cellular responses in primigravidae (G1), women in their second to fourth pregnancy (G2-4) and grand multigravidae we sought to identify key components of the dysregulated immune response. PBMC were exposed to CS2-infected erythrocytes (IE) opsonised with autologous plasma or unopsonised IE, and cytokine and chemokine secretion was measured. Higher levels of opsonising antibody were present in plasma derived from multigravid compared to primigravid women. Significant differences in the levels of cytokines and chemokines secreted in response to IE were observed. Less IL-10, IL-1ß, IL-6 and TNF but more CXCL8, CCL8, IFNγ and CXCL10 were detected in G5-7 compared to G2-4 women. Our study provides fresh insight into the modulation of peripheral blood cell function and effects on the balance between host protection and immunopathology during placental malaria infection.

Assessment of antibody-dependent respiratory burst activity from mouse neutrophils on Plasmodium yoelii malaria challenge outcome.

New tools are required to expedite the development of an effective vaccine against the blood-stage infection with the human malaria parasite Plasmodium falciparum. This work describes the assessment of the ADRB assay in a mouse model, characterizing the functional interaction between antimalarial serum antibodies and FcRs upon neutrophils. We describe a reproducible, antigen-specific assay, dependent on functional FcR signaling, and show that ADRB activity is induced equally by IgG1 and IgG2a isotypes and is modulated by blocking FcR function. However, following immunization of mice with the blood-stage vaccine candidate antigen MSP142, no measurable ADRB activity was induced against PEMS and neither was vaccine efficacy modulated against Plasmodium yoelii blood-stage challenge in γ(-/-) mice compared with WT mice. In contrast, following a primary, nonlethal P. yoelii parasite challenge, serum from vaccinated mice and nonimmunized controls showed anti-PEMS ADRB activity. Upon secondary challenge, nonimmunized γ(-/-) mice showed a reduced ability to control blood-stage parasitemia compared with immunized γ(-/-) mice; however, WT mice, depleted of their neutrophils, did not lose their ability to control infection. Thus, whereas neutrophil-induced ADRB against PEMS does not appear to play a role in protection against P. yoelii rodent malaria, induction of ADRB activity after challenge suggests that antigen targets of anti-PEMS ADRB activity remain to be established, as well as further supporting the observation that ADRB activity to P. falciparum arises following repeated natural exposure.

The utility of Plasmodium berghei as a rodent model for anti-merozoite malaria vaccine assessment.

Rodent malaria species Plasmodium yoelii and P. chabaudi have been widely used to validate vaccine approaches targeting blood-stage merozoite antigens. However, increasing data suggest the P. berghei rodent malaria may be able to circumvent vaccine-induced anti-merozoite responses. Here we confirm a failure to protect against P. berghei, despite successful antibody induction against leading merozoite antigens using protein-in-adjuvant or viral vectored vaccine delivery. No subunit vaccine approach showed efficacy in mice following immunization and challenge with the wild-type P. berghei strains ANKA or NK65, or against a chimeric parasite line encoding a merozoite antigen from P. falciparum. Protection was not improved in knockout mice lacking the inhibitory Fc receptor CD32b, nor against a Δsmac P. berghei parasite line with a non-sequestering phenotype. An improved understanding of the mechanisms responsible for protection, or failure of protection, against P. berghei merozoites could guide the development of an efficacious vaccine against P. falciparum.

T cell responses induced by adenoviral vectored vaccines can be adjuvanted by fusion of antigen to the oligomerization domain of C4b-binding protein.

Viral vectored vaccines have been shown to induce both T cell and antibody responses in animals and humans. However, the induction of even higher level T cell responses may be crucial in achieving vaccine efficacy against difficult disease targets, especially in humans. Here we investigate the oligomerization domain of the α-chain of C4b-binding protein (C4 bp) as a candidate T cell "molecular adjuvant" when fused to malaria antigens expressed by human adenovirus serotype 5 (AdHu5) vectored vaccines in BALB/c mice. We demonstrate that i) C-terminal fusion of an oligomerization domain can enhance the quantity of antigen-specific CD4(+) and CD8(+) T cell responses induced in mice after only a single immunization of recombinant AdHu5, and that the T cells maintain similar functional cytokine profiles; ii) an adjuvant effect is observed for AdHu5 vectors expressing either the 42 kDa C-terminal domain of Plasmodium yoelii merozoite surface protein 1 (PyMSP1(42)) or the 83 kDa ectodomain of P. falciparum strain 3D7 apical membrane antigen 1 (PfAMA1), but not a candidate 128kDa P. falciparum MSP1 biallelic fusion antigen; iii) following two homologous immunizations of AdHu5 vaccines, antigen-specific T cell responses are further enhanced, however, in both BALB/c mice and New Zealand White rabbits no enhancement of functional antibody responses is observed; and iv) that the T cell adjuvant activity of C4 bp is not dependent on a functional Fc-receptor γ-chain in the host, but is associated with the oligomerization of small (<80 kDa) antigens expressed by recombinant AdHu5. The oligomerization domain of C4 bp can thus adjuvant T cell responses induced by AdHu5 vectors against selected antigens and its clinical utility as well as mechanism of action warrant further investigation.

Recombinant viral-vectored vaccines expressing Plasmodium chabaudi AS apical membrane antigen 1: mechanisms of vaccine-induced blood-stage protection.

Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.

Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference.

Replication-deficient adenovirus and modified vaccinia virus Ankara (MVA) vectors expressing single pre-erythrocytic or blood-stage Plasmodium falciparum Ags have entered clinical testing using a heterologous prime-boost immunization approach. In this study, we investigated the utility of the same immunization regimen when combining viral vectored vaccines expressing the 42-kDa C terminus of the blood-stage Ag merozoite surface protein 1 and the pre-erythrocytic Ag circumsporozoite protein in the Plasmodium yoelii mouse model. We find that vaccine coadministration leads to maintained Ab responses and efficacy against blood-stage infection, but reduced secondary CD8(+) T cell responses against both Ags and efficacy against liver-stage infection. CD8(+) T cell interference can be minimized by coadministering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy in mice immunized against both Ags compared with just one. CD8(+) T cell interference (following MVA coadministration as a mixture) may be caused partly by a lack of physiologic space for high-magnitude responses against multiple Ags, but is not caused by competition for presentation of Ag on MHC class I molecules, nor is it due to restricted T cell access to APCs presenting both Ags. Instead, enhanced killing of peptide-pulsed cells is observed in mice possessing pre-existing T cells against two Ags compared with just one, suggesting that priming against multiple Ags may in part reduce the potency of multiantigen MVA vectors to stimulate secondary CD8(+) T cell responses. These data have important implications for the development of a multistage or multicomponent viral vectored malaria vaccine for use in humans.

The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus.

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.