CLN8 disease caused by large genomic deletions

BACKGROUND: The presence of deletions can complicate genetic diagnosis of autosomal recessive disease. METHOD: The DNA of patients was analyzed in a diagnostic setting. RESULTS: We present three unrelated patients each carrying deletions that encompass the 37 kb CLN8 gene and discuss their phenotype. Two of the cases were hemizygous for a mutant allele - their deletions unmasked a mutation in CLN8 on the other chromosome. CONCLUSION: Microarray analysis is recommended in any patient suspected of NCL who is apparently homozygous for a mutation that is not present in one of the parents or when the family has no known consanguinity

Development of statistical methods for the analysis of single-cell RNA-seq data

Single-cell RNA-sequencing profiles the transcriptome of cells from diverse populations. A popular intermediate data format is a large count matrix of genes x cells. This type of data brings several analytical challenges. Here, I present three projects that I worked on during my PhD that address particular aspects of working with such datasets: - The large number of cells in the count matrix is a challenge for fitting gamma-Poisson generalized linear models with existing tools. I developed a new R package called glmGamPoi to address this gap. I optimized the overdispersion estimation procedure to be quick and robust for datasets with many cells and small counts. I compared the performance against two popular tools (edgeR and DESeq2) and find that my inference is 6x to 13x faster and achieves a higher likelihood for a majority of the genes in four single-cell datasets. - The variance of single-cell RNA-seq counts depends on their mean but many existing statistical tools have optimal performance when the variance is uniform. Accordingly, variance-stabilizing transformations are applied to unlock the large number of methods with such an requirement. I compared four approaches to variance-stabilize the data based on the delta method, model residuals, inferred latent expression state or count factor analysis. I describe the theoretical strength and weaknesses, and compare their empirical performance in a benchmark on simulated and real single-cell data. I find that none of the mathematically more sophisticated transformations consistently outperform the simple log(y/s+1) transformation. - Multi-condition single-cell data offers the opportunity to find differentially expressed genes for individual cell subpopulations. However, the prevalent approach to analyze such data is to start by dividing the cells into discrete populations and then test for differential expression within each group. The results are interpretable but may miss interesting cases by (1) choosing the cluster size too small and lacking power to detect effects or (2) choosing the cluster size too large and obscuring interesting effects apparent on a smaller scale. I developed a new statistical framework for the analysis of multi-condition single-cell data that avoids the premature discretization. The approach performs regression on the latent subspaces occupied by the cells in each condition. The method is implemented as an R package called lemur

CMDX©-based single source information system for simplified quality management and clinical research in prostate cancer

Background: Histopathological evaluation of prostatectomy specimens is crucial to decision-making and prediction of patient outcomes in prostate cancer (PCa). Topographical information regarding PCa extension and positive surgical margins (PSM) is essential for clinical routines, quality assessment, and research. However, local hospital information systems (HIS) often do not support the documentation of such information. Therefore, we investigated the feasibility of integrating a cMDX-based pathology report including topographical information into the clinical routine with the aims of obtaining data, performing analysis and generating heat maps in a timely manner, while avoiding data redundancy. Methods: We analyzed the workflow of the histopathological evaluation documentation process. We then developed a concept for a pathology report based on a cMDX data model facilitating the topographical documentation of PCa and PSM; the cMDX SSIS is implemented within the HIS of University Hospital Muenster. We then generated a heat map of PCa extension and PSM using the data. Data quality was assessed by measuring the data completeness of reports for all cases, as well as the source-to-database error. We also conducted a prospective study to compare our proposed method with recent retrospective and paper-based studies according to the time required for data analysis. Results: We identified 30 input fields that were applied to the cMDX-based data model and the electronic report was integrated into the clinical workflow. Between 2010 and 2011, a total of 259 reports were generated with 100% data completeness and a source-to-database error of 10.3 per 10,000 fields. These reports were directly reused for data analysis, and a heat map based on the data was generated. PCa was mostly localized in the peripheral zone of the prostate. The mean relative tumor volume was 16.6%. The most PSM were localized in the apical region of the prostate. In the retrospective study, 1623 paper-based reports were transferred to cMDX reports; this process took 15 ± 2 minutes per report. In a paper-based study, the analysis data preparation required 45 ± 5 minutes per report. Conclusions: cMDX SSIS can be integrated into the local HIS and provides clinical routine data and timely heat maps for quality assessment and research purposes.

Variability of Saliva Viscosity - Potential Impact

As novel COVID-19 testing develops, saliva has become of increasing interest as an alternate biological sample for rapid testing. The appeal in saliva-based testing lies within the ease of which samples are collected, as well as patient comfort throughout the collection process. With this, it has become increasingly important to delineate the characteristics of saliva viscosity due to its effects on the movement and interactions of the substances and molecules found within it. The characteristics that affect saliva viscosity include the presence of aggregates, variations in temperature, and time elapsed between sample collection and testing. Understanding how physicochemical properties and temperature affect saliva’s viscosity are important in generating guidelines for proper sample handling in saliva testing to ensure consistent and reliable results. In this study, passive sampling of saliva was analyzed. This type of collection ensures a more uniform saliva composition, suggesting that variations in viscosity can be attributed solely to modifications in saliva handling post-collection. The data suggested that saliva viscosity is greatest immediately following collection of the saliva sample, increases with higher quantities of aggregates in saliva, and decreases tremendously when the sample has been frozen and thawed to room temperature. These findings suggest that to ensure accuracy and uniformity in quantitative saliva-based test results, protocols should favor the testing of a sample immediately following its collection. The implications of these results in optimizing saliva testing are far reaching. The value of saliva based testing extends far beyond COVID-19 or other disease testing. It is also gaining utility in understanding daily fluctuations in hydration state and in other wellness applications

Epilepsy in infancy study: a population based study on epilepsies with onset in the first two years of life

Children with the onset of epilepsy under the age of two years are a heterogeneous group with particularly poor outcome reported by studies mostly originating from specialist hospital / clinic settings. A population based study was designed to determine the incidence of epilepsy onset under the age of two years and to ascertain a cohort for long-term follow up. This cohort observation aimed to determine usefulness of the current international classification of epilepsies in this age group, associated structural brain abnormalities, predictors of developmental function close to diagnosis; and factors that are associated with a longitudinal change of developmental scores after 12 months. Cases were prospectively identified from residents in 15 boroughs of North London involving hospital and community based paediatricians. Information was obtained either by questionnaire anonymously or from clinical assessments of children enrolled in the observational cohort. 57 cases were enrolled giving a crude annual incidence of 54 (95% CI 41-69) /100.000 children under the age of 2 years. A two source capture recapture model determined an adjusted annual incidence of 56.3 – 88.5 (95% CI) / 100.000 <= 2 years. Clinical assessment of children in the observational cohort at baseline and 12 months follow up included neurodevelopmental evaluation using standardised tools, central review of EEG’s and neuroimages. Data of all subjects enrolled were independently evaluated by two paediatric neurologists. Although most cases were classified under epilepsy syndrome groups, a specific epilepsy syndrome diagnosis could not be allocated in a third of cases and there was only moderate inter-rater agreement (kappa scores: 0.48, 0.5). Review of neuroimages of 52 children (91%) demonstrated a high yield of aetiologically relevant abnormalities in 26 (50%) with common occurrence of developmental malformations identified in 11 (43%). Multivariate regression analysis showed that 'abnormal neurological examination' and 'presence of interictal discharges on EEG' significantly and independently predicted lower developmental function close to diagnosis. There was no significant difference between initial and developmental function on follow up after adjusting for initial infantile spasms, normal or abnormal initial EEG, seizure status at follow up, structural brain abnormalities, and antiepileptic medication taken, suggesting that the initial status determined the function after 12 months follow up

BBF RFC 105: The Intein standard - a universal way to modify proteins after translation

This Request for Comments (RFC) proposes a new standard that allows for easy and flexible cloning of intein constructs and thus makes this technology accessible to the synthetic biology community

Impact of HPV Testing Based on the 2020 Update of the German Cervical Cancer Screening Program—Data from a Retrospective Monocentric Study

Background/Objectives: Since 2020, Germany has replaced its annual cytological screening for cervical cancer in women aged 35 and older with a combination screening program. This updated protocol, conducted every three years, includes both a cervical Pap smear and an HPV (human papillomavirus) test. In addition, the 2020 update of the German Cervical Cancer Screening Program mandates a more timely follow-up for certain abnormal findings compared to the previous approach. Methods: Results of cytologic, colposcopic, and histologic examinations between 2018 and 2021 were retrospectively retrieved from the electronic patient records of the Institute of Pathology Saarbrücken Rastpfuhl in order to present relevant findings within the years 2018–2019 and 2020–2021. The present study included all women who received appropriate follow-up based on the corresponding underlying screening modality. Results: Our reporting considered data from 30,715 women in 2018/19 (prior screening modality group) and 25,924 women in 2020/21 (updated screening modality group). In 2018/19, a total of 93 histological examinations were performed, compared to 237 in 2020/21. In 2018/19, 52 cases of CIN III and 12 cases of CIN II were identified, while in 2020/21, 78 cases of CIN III and 31 cases of CIN II were detected. In contrast, while women with negative cytological findings in 2018/19 were typically not referred for further colposcopic or histological evaluation, the updated program enabled earlier detection and treatment of cases with diagnosed high-grade dysplasia based solely on a positive HPV test. Notably, 31 women who were diagnosed at an earlier stage based solely on a positive HPV test initially presented with a cytological Pap I result (negative for intraepithelial lesion or malignancy; NILM). Conclusions: Additional HPV co-testing within the updated German cervical cancer screening program resulted not only in high rates of high-grade dysplasia detection but also a rise in the number of colposcopic procedures. While negative follow-up HPV findings regularly showed remission of the original finding, incidence of high-grade dysplasia was usually linked to a positive HPV test

Stereo-EEG exploration in the insula/operculum in paediatric patients with refractory epilepsy

PURPOSE: Failure to recognise involvement of the insula / opercula (I/O) region is associated with poor outcome in epilepsy surgery. Recognition is challenging due to high connectivity with adjacent structures resulting in variable and misleading semiology, often subjective and therefore likely to be underreported by children. In this study we explored prevalence and characteristics of I/O involvement in paediatric patients undergoing sEEG exploration. METHOD: We retrospectively included all consecutive patients undergoing sEEG at our centre between 11/2014 and 01/2018 with at least three contacts within I/O and excluded those with undetermined seizure onset zone (SOZ) by sEEG. We divided patients into three groups: 1) SOZ in I/O, 2) spread to I/O and 3) no I/O involvement. We compared pre-invasive characteristics, sEEG results, surgery and outcome for each group. RESULTS: 29 of all 53 consecutive patients had an identified SOZ by sEEG and at least three contacts within the I/O and were included. 41% had I/O SOZ, 38% had I/O spread and 21% had no I/O involvement. Insula associated symptoms described in adult literature were not statistically different between the three groups. Complications due to sEEG were low (2 of 53 patients). Following I/O surgery, 63% were seizure free while an additional 26% of patients achieved seizure reduction. Postoperative deficits were seen in 75% of the patients but completely resolved in all but one patient. CONCLUSIONS: Our data suggest an important role of the I/O region with frequent onset or propagation to the I/O region (at least 64% of all 53 sEEG cases). Semiology appears less specific than in adults. Insula depth electrode insertion is safe with subsequent good surgical outcomes albeit common transient deficits

Results of an international Delphi consensus in epilepsy with myoclonic atonic seizures/ Doose syndrome

OBJECTIVE: To establish a standard framework for early phenotypic diagnosis, investigations, expected findings from investigations, evolution, effective therapies and prognosis in the syndrome of Epilepsy with myoclonic atonic seizures (EMAS) / Doose syndrome. METHODS: A core study group (CSG) interested in EMAS was convened. CSG then identified and nominated 15 experts in the field of EMAS. This expert panel (EP) from English speaking nations was invited to participate in anonymous questionnaires. A literature review was provided to them (supplement 1). Three rounds of questionnaires were sent to identify areas of consensus, strength of consensus and areas of contention. RESULTS: Strong consensus was obtained regarding the clinical phenotype of EMAS: myoclonic atonic seizure was identified among others as a mandatory seizure type with typical onset of afebrile seizures between one and six years. A new term "stormy phase" (SP) was designated to delineate a characteristic phenotypic evolution in EMAS patients associated with seizure worsening. Strong consensus regarding the existence and time of onset of the SP, mandatory investigations to be performed early and later in the clinical course of EMAS, first and second tier treatment and prognostic factors for poor outcome were identified. Areas of lack of consensus included some seizure types that are necessary to diagnose EMAS, interictal EEG findings that prognosticate the course of EMAS, overall duration of SP, time to complete remission, and best approach to treat drug resistant EMAS. SIGNIFICANCE: Expert consensus on core diagnostic criteria of EMAS necessary for natural history studies, phenotype-genotype correlations, and clinical trials including comparative studies was demonstrated. Areas of disagreements (especially prognostic features; treatment options) need further research

Dietary Management of Children With Super-Refractory Status Epilepticus: A Systematic Review and Experience in a Single UK Tertiary Centre

Ketogenic diet therapies (KDT) are high-fat, low carbohydrate diets used as an effective treatment option for drug-resistant epilepsy. There is limited research on the efficacy of KDT for super-refractory status epilepticus (SRSE). We systematically review evidence for use of KDT in children with SRSE and present a single UK tertiary centre's experience. Thirty one articles were included, of which 24 were “medium” or “low” quality. One hundred and forty seven children with SRSE started KDT, of which 141 (96%) achieved ketosis. KDT was started mean 5.3 days (range 1–420) after status epilepticus (SE) started. SRSE resolved in 85/141 (60%) children after mean 6.3 days (range 0–19) post SE onset, but it is unclear whether further treatments were initiated post-KDT. 13/141 (9%) children died. Response to KDT was more likely when initiated earlier (p = 0.03) and in females (p = 0.01). Adverse side effects were reported in 48/141 (34%), mostly gastrointestinal; potentially serious adverse effects occurred in ≤4%. Eight children with SRSE, all diagnosed with febrile infection-related epilepsy syndrome, were treated with KDT at Great Ormond Street Hospital for Children. KDT was initiated enterally at mean day 13.6+/− 5.1 of admission. Seven of 8 (88%) children reported adverse side effects, which were potentially serious in 4/8 (50%), including metabolic acidosis, hypoglycaemia and raised amylase. SE ceased in 6/8 (75%) children after mean 25+/− 9.4 days post onset, but other treatments were often started concomitantly and all children started other treatments post-KDT. Two of 8 (25%) children died during admission and another died post-admission. Four of the remaining 5 children continue to have drug-resistant seizures, one of whom remains on KDT; seizure burden was unknown for one child. Our findings indicate that KDT is possible and safe in children with SRSE. Cessation of SRSE may occur in almost two-thirds of children initiated with KDT, but a causal effect is difficult to determine due to concomitant treatments, treatments started post-KDT and the variable length of time post-KDT onset when SRSE cessation occurs. Given that serious adverse side effects seem rare and response rates are (cautiously) favorable, KDT should be considered as an early treatment option in this group