Pitfalls and challenges with population assignments of individuals from admixed populations: Applying Genogeographer on Brazilian individuals.

The assignment of individuals to a population can be of importance for the identification of mass disaster victims or criminal offenders in the field of forensic genetics. This assignment is based on biostatistical methods that process data of ancestry informative markers (AIMs), which are selected based on large allele frequency differences between the populations of interest. However, population assignments of individuals with an admixed genetic background are challenging. Admixed individuals are genetic mosaics of chromosomal segments from the parental populations, which may lead to ambiguous or no population assignment. This is problematic since admixture events are a substantial part of human history. In this study, we present challenges of interpreting the evidential weight of population assignments. We used Genogeographer for likelihood ratio (LR) calculations and Brazilians as examples of admixed individuals. Brazilians are a very heterogenous population representing a three-way admixture between Native Americans, Europeans, and Africans. Ancestry informative markers were typed in a total of 589 individuals from Brazil using the Precision ID Ancestry Panel. The Brazilians were assigned to six metapopulations (East Asia, Europe, Middle East, North Africa, South-Central Asia, Sub-Saharan Africa) defined in the Genogeographer software and LRs were calculated if the AIM profile was not an outlier in all metapopulations and simulated two-way (1:1) admixtures of the six metapopulations. Population assignments failed for 55% of the samples. These samples had significantly higher genetic contributions from East Asia, South-Central Asia and Sub-Saharan Africa, and significantly lower genetic contributions from Europe. Most of the individuals with population assignments were assigned to the metapopulations of Middle East (58%) or North Africa (36%), followed by Europe (4%), South-Central Asia (1%), and Sub-Saharan Africa (1%). For 8% of the samples, population assignments were only possible when assignments to simulated two-way (1:1) admixtures of the six metapopulations were considered. Most of these individuals were assigned to two-way admixtures of North Africa, South-Central Asia, or Sub-Saharan Africa. Relatively low median likelihood ratios (LRs<1000) were observed when comparing population likelihoods for Europe, Middle East, North Africa, South-Central Asia, or simulated 1:1 admixtures of these metapopulations. Comparisons including East Asian or Sub-Saharan African populations resulted in larger median LRs (LR>1010). The results suggested that the Precision ID Ancestry Panel provided too little information and that additional markers specifically selected for sub-continental differentiation may be required for accurate population assignment of admixed individuals. Furthermore, a Genogeographer database with additional populations including admixed populations would be advantageous for interpretation of admixed AIM profiles. It would likely increase the number of population assignments and illustrate alternatives to the most likely population, which would be valuable information for the case officer when writing the case report.

How does cell-based non-invasive prenatal test (NIPT) perform against chorionic villus sampling and cell-free NIPT in detecting trisomies and copy number variations? A clinical study from Denmark.

OBJECTIVES: We aimed to compare cell-based NIPT (cbNIPT) to chorionic villus sampling (CVS) and to examine the test characteristics of cbNIPT in the first clinical validation study of cbNIPT compared to cell-free NIPT (cfNIPT). MATERIAL AND METHODS: Study 1: Women (N = 92) who accepted CVS were recruited for cbNIPT (53 normal and 39 abnormal). Samples were analyzed with chromosomal microarray (CMA). Study 2: Women (N = 282) who accepted cfNIPT were recruited for cbNIPT. cfNIPT was analyzed using sequencing and cbNIPT by CMA. RESULTS: Study 1: cbNIPT detected all aberrations (32/32) found in CVS: trisomies 13, 18 and 21 (23/23), pathogenic copy number variations (CNVs) (6/6) and sex chromosome aberrations (3/3). cbNIPT detected 3/8 cases of mosaicism in the placenta. Study 2: cbNIPT detected all trisomies found with cfNIPT (6/6) and had no false positive (0/246). One of the three CNVs called by cbNIPT was confirmed by CVS but was undetected by cfNIPT, two were false positives. cbNIPT detected mosaicism in five samples, of which two were not detected by cfNIPT. cbNIPT failed in 7.8% compared to 2.8% in cfNIPT. CONCLUSION: Circulating trophoblasts in the maternal circulation provide the potential of screening for aneuploidies and pathogenic CNVs covering the entire fetal genome.

Cytosolic phosphofructokinases are important for sugar homeostasis in leaves of Arabidopsis thaliana.

BACKGROUND AND AIMS: ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focuses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana. METHODS: We isolated homozygous knockout individual mutants (pfk1, pfk3, pfk6 and pfk7) and two double mutants (pfk1/7 and pfk3/6), and characterized their growth and metabolic phenotypes. KEY RESULTS: In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and showed a clear visual and metabolic phenotype with reduced shoot growth, early flowering and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates. CONCLUSIONS: We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently in source-sink relationships in A. thaliana, while PFK3 and PFK6 only play a minor role under normal growth conditions.

The Circadian Clock Gene Circuit Controls Protein and Phosphoprotein Rhythms in Arabidopsis thaliana.

Twenty-four-hour, circadian rhythms control many eukaryotic mRNA levels, whereas the levels of their more stable proteins are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and phosphoproteomic time series from Arabidopsis thaliana plants under constant light conditions, estimating that just 0.4% of quantified proteins but a much larger proportion of quantified phospho-sites were rhythmic. Approximately half of the rhythmic phospho-sites were most phosphorylated at subjective dawn, a pattern we term the "phospho-dawn." Members of the SnRK/CDPK family of protein kinases are candidate regulators. A CCA1-overexpressing line that disables the clock gene circuit lacked most circadian protein phosphorylation. However, the few phospho-sites that fluctuated despite CCA1-overexpression still tended to peak in abundance close to subjective dawn, suggesting that the canonical clock mechanism is necessary for most but perhaps not all protein phosphorylation rhythms. To test the potential functional relevance of our datasets, we conducted phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase/phosphatase (F2KP), as an example. The rhythmic phosphorylation of diverse protein targets is controlled by the clock gene circuit, implicating posttranslational mechanisms in the transmission of circadian timing information in plants.

Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing.

PURPOSE: Proof of concept of the use of cell-based non-invasive prenatal testing (cbNIPT) as an alternative to chorionic villus sampling (CVS) following preimplantation genetic testing for monogenic disorders (PGT-M). METHOD: PGT-M was performed by combined testing of short tandem repeat (STR) markers and direct mutation detection, followed by transfer of an unaffected embryo. Patients who opted for follow-up of PGT-M by CVS had blood sampled, from which potential fetal extravillous throphoblast cells were isolated. The cell origin and mutational status were determined by combined testing of STR markers and direct mutation detection using the same setup as during PGT. The cbNIPT results with respect to the mutational status were compared to those of genetic testing of the CVS. RESULTS: Eight patients had blood collected between gestational weeks 10 and 13, from which 33 potential fetal cell samples were isolated. Twenty-seven out of 33 isolated cell samples were successfully tested (82%), of which 24 were of fetal origin (89%). This corresponds to a median of 2.5 successfully tested fetal cell samples per case (range 1-6). All fetal cell samples had a genetic profile identical to that of the transferred embryo confirming a pregnancy with an unaffected fetus, in accordance with the CVS results. CONCLUSION: These findings show that although measures are needed to enhance the test success rate and the number of cells identified, cbNIPT is a promising alternative to CVS. TRIAL REGISTRATION NUMBER: N-20180001.

PAX2 variant associated with bilateral kidney agenesis and broad intrafamilial disease variability.

Pathogenic variants in PAX2 have previously been associated with renal coloboma syndrome. Here we present a novel variant c.68T>C associated with bilateral kidney agenesis, minimal change nephropathy, ureteropelvic junction obstruction, duplex kidney with hydronephrosis of upper pole system and bilateral kidney hypoplasia within the same family. Additionally, two family members were found to have optic nerve abnormalities further supporting the impact of the PAX2 variant. This is the first report of a PAX2 variant associated with bilateral kidney agenesis.

Development of an automated AmpliSeq™ library building workflow for biological stain samples on the Biomek® 3000.

Here, we present the development of an automated AmpliSeq™ (ThermoFischer, MA, USA) workflow for library building using the Biomek® 3000 Laboratory Automation Workstation (Beckman Coulter Inc., CA, USA), in which the total volume of PCR reagents and reagents for library preparation are reduced by one-half. The automated AmpliSeq workflow was tested using 43 stain samples (blood, bone, muscle tissue, semen, swab, nail scrape and cigarette butts) collected from crime scenes. The sequencing data were evaluated for locus balance, heterozygous allele balance and noise. The performance of libraries built with the automated AmpliSeq workflow using one-half of the recommended reagent volumes were similar to the performance of libraries built with the recommended (full) volumes of the reagents.

Ancestry prediction efficiency of the software GenoGeographer using a z-score method and the ancestry informative markers in the Precision ID Ancestry Panel.

We compared the efficiency of the freely available software GenoGeographer that includes a z-score based analysis with that of a naïve method based on the maximal likelihoods of 164 of the 165 ancestral informative markers (AIM) that are included in the commercially available kit Precision ID Ancestry Panel from Thermo Fisher Scientific. The AIM profiles were obtained by investigations with the Precision ID Ancestry Panel in our laboratory and from SNP data in the literature and publically available databases. We established eight well-defined AIM reference population data sets from 3603 AIM profiles. Six reference populations with profiles from multiple populations (Sub-Saharan Africa, North Africa, Middle East, Europe, South/Central Asia, East Asia), and two populations with individuals with admixed ancestry (Somalia and Greenland). By means of GenoGeographer and naïve calculations of the maximal likelihoods, 566 AIM profiles from individuals that were not included in the reference populations and expected to belong to one of the eight reference populations were tested. An initial standard z-score based test with GenoGeographer demonstrated that 22.4% of the individuals could not be assigned to any of the reference populations. Among the remaining 77.6% of the individuals, 83.6% were assigned to the reference population that was concordant with the specified populations of origin of the individuals, 8.2% had ambiguous assignments because they could belong to both the specified population of origin and one or more of the other populations, and 8.2% were assigned to a reference population that was discordant from the specified population of origin. A naïve assignment based on the maximal likelihood resulted in 78.1% concordant and 21.9% discordant assignments. The results demonstrate that the z-score analysis with GenoGeographer can reduce the error rate with a factor of almost three compared with that of the naïve estimation based on the maximal likelihoods of the AIM profiles. The Precision ID Ancestry Panel is a useful kit for the assignment of ancestry of the eight investigated populations that included two admixed populations. More AIMs with better discrimination and more data on the distribution of AIMs in relevant populations are needed to improve the efficiency of genogeographic prediction with AIMs on a worldwide basis.

Enzyme Replacement Therapy During Pregnancy in Fabry Patients : Review of Published Cases of Live Births and a New Case of a Severely Affected Female with Fabry Disease and Pre-eclampsia Complicating Pregnancy.

Fabry disease (FD) is an X-linked, lysosomal storage disease. Mutations in the gene coding for alpha-galactosidase A lead to globotriaosylceramide (Gb-3) accumulation in lysosomes and in placenta and umbilical cord. Impact of FD and treatment with enzyme replacement (ERT) on foetal development is undisclosed.A 38-year-old primigravida with FD (G85N) is reported. She has 50% reduced alpha-galactosidase A activity and elevated plasma and urine-Gb-3. She was severely affected with ischaemic stroke at age 23, hypertension, albuminuria and moderately reduced renal function. ERT was initiated at age 23 years in 2001 and continued during spontaneous pregnancy at age 38. In third trimester she developed moderate-to-severe pre-eclampsia, successfully managed by methyldopa. Chorion villus sampling revealed a male foetus without the maternal gene mutation. Planned Caesarean section was performed without complications at gestational age week 38 + 6, delivering a healthy boy. Histopathological placental examination showed no sign of Gb-3 accumulation. Literature survey disclosed a total of 12 cases, 8 were treated with ERT during pregnancy and 5 infants inherited the family mutation. All outcomes were successful. In the six cases with available placental histopathological examination, Gb-3 accumulation was only seen on the foetal side if the foetus had the inherited mutation.In conclusion, the present case, describing the first data from a severely affected FD patient receiving ERT during pregnancy complicated by pre-eclampsia, together with all other published cases, has emphasized that ERT is safe during pregnancy and resulting in successful foetal outcome; despite this, ERT is by the health authorities advised against during pregnancy.

Sequencing of 231 forensic genetic markers using the MiSeq FGx™ forensic genomics system - an evaluation of the assay and software.

The MiSeq FGx™ Forensic Genomics System types 231 genetic markers in one multiplex polymerase chain reaction (PCR) assay. The markers include core forensic short tandem repeats (STRs) as well as identity, ancestry and phenotype informative short nucleotide polymorphisms (SNPs). In this work, the MiSeq FGx™ Forensic Genomics System was evaluated by analysing reproducibility, sensitivity, mixture identification and forensic phenotyping capabilities of the assay. Furthermore, the genotype calling of the ForenSeq™ Universal Analysis Software was verified by analysing fastq.gz files from the MiSeq FGx™ platform using the softwares STRinNGS and GATK. Overall, the performance of the MiSeq FGx™ Forensic Genomics System was high. However, locus and allele drop-outs were relatively frequent at six loci (two STRs and four human identification SNPs) due to low read depth or skewed heterozygote balances, and the stutter ratios were larger than those observed with conventional STR genotyping methods. The risk of locus and allele drop-outs increased dramatically when the amount of DNA in the first PCR was lower than 250 pg. Two-person 50:1 mixtures were identified as mixtures, whereas 100:1 and 1 000:1 mixtures were not. Y-chromosomal short tandem repeats (Y-STRs) alleles were detected in the 100:1 and 1 000:1 female/male mixtures. The ForenSeq™ Universal Analysis Software provided the data analyst with useful alerts that simplified the analysis of the large number of markers. Many of the alerts were due to user-defined, locus-specific criteria. The results shown here indicated that the default settings should be altered for some loci. Also, recommended changes to the assay and software are discussed.

Stutter analysis of complex STR MPS data.

Stutters are common and well documented artefacts of amplification of short tandem repeat (STR) regions when using polymerase chain reaction (PCR) occurring as strands one or more motifs shorter or longer than the parental allele. Understanding the mechanism and rate by which stutters are created is especially important when the samples contain small amounts of DNA or DNA from multiple contributors. It has been shown that there is a linear relationship between the longest uninterrupted stretch (LUS) and the stutter ratio. This holds if there is only a single type of stutter variant. However, with massively parallel sequencing (MPS), we see that alleles may create different stutters corresponding to stuttering of different parts of the parental allele. This calls for a refinement of the LUS concept. We analysed all uninterrupted stretches, here called blocks, and identified the block from which the stutter originated. We defined the block length of the missing motif (BLMM) as the length of the identified block. We found that the relationship between the stutter ratio and BLMM was linear using a simple system of recurrence relations. We found that the mean square error decreased by a factor up to 17.5 for compound and complex autosomal markers when using BLMM instead of LUS.

Quantification of massively parallel sequencing libraries - a comparative study of eight methods.

Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out.

Weight of the evidence of genetic investigations of ancestry informative markers.

Ancestry-informative markers (AIMs) are markers that give information about the ancestry of individuals. They are used in forensic genetics for predicting the geographic origin of the investigated individual in crime and identification cases. In the exploration of the genogeographic origin of an AIMs profile, the likelihoods of the AIMs profile in various populations may be calculated. However, there may not be an appropriate reference population in the database. The fact that the likelihood ratio (LR) of one population compared to that of another population is large does not imply that any of the populations is relevant. To handle this phenomena, we derived a likelihood ratio test (LRT) that is a measure of absolute concordance between an AIMs profile and a population rather than a relative measure of the AIMs profile's likelihood in two populations. The LRT is similar to a Fisher's exact test. By aggregating over markers, the central limit theorem suggests that the resulting quantity is approximately normally distributed. If only a few markers are genotyped or if the majority of the markers are fixed in a given population, the approximation may fail. We overcome this using importance sampling and show how exponential tilting results in an efficient proposal distribution. By simulations and published AIMs profiles, we demonstrate the applicability of the derived methodology. For the genotyped AIMs, the LRT approach achieves the nominal levels of rejection when tested on data from five major continental regions.

YfilerⓇ Plus population samples and dilution series: stutters, analytic thresholds, and drop-out probabilities.

The Yfiler Ⓡ Plus Amplification Kit amplifies 27 Y chromosomal small tandem repeat (STR) markers. The kit has five-fluorescent dye chemistry and the improved PCR buffer system of modern STR kits. We validated the kit for accredited investigations of crime scene samples by a thorough study of kit dynamics and performance. We determined dye-dependent analytical thresholds by receiver operating characteristics (ROC) and made a customised artefact filter that includes theoretical known artefacts by use of previously analysed population samples. Dilution series of known male DNA and a selection of crime scene samples were analysed with the customised thresholds and artefact filters. The Yfiler Ⓡ Plus Amplification Kit was sensitive giving full profiles down to 70 pg of male DNA. The balances between the fluorescent dyes as well as between loci were very good. The kit was able to produce full Y-STR profiles from crime scene samples containing small amounts of male DNA and large amounts of female DNA (although unspecific reactions were evident for very unbalanced mixtures). A decrease in the drop-out rate was found for both the dilution series and population samples, as well as a small increase in the drop-in rate for population samples, using the customised threshold and artefact filters compared to company-provided thresholds and artefact filters. The additional drop-ins were all of a nature that would be detected by inspection of the results. For the crime scene samples, large amounts of female DNA complicated the analysis by causing drop-ins of characteristic female DNA artefacts. Even though the customised analytical threshold in combination with the custom-made artefact filters gave more alleles, crime scene samples still needed special attention from the forensic geneticist.

Evaluation of the Precision ID Ancestry Panel for crime case work: A SNP typing assay developed for typing of 165 ancestral informative markers.

The application of massive parallel sequencing (MPS) methodologies in forensic genetics is promising and it is gradually being implemented in forensic genetic case work. One of the major advantages of these technologies is that several traditional electrophoresis assays can be combined into one single MPS assay. This reduces both the amount of sample used and the time of the investigations. This study assessed the utility of the Precision ID Ancestry Panel (Thermo Fisher Scientific, Waltham, USA) in forensic genetics. This assay was developed for the Ion Torrent PGM™ System and genotypes 165 ancestry informative SNPs. The performance of the assay and the accompanying software solution for ancestry inference was assessed by typing 142 Danes and 98 Somalis. Locus balance, heterozygote balance, and noise levels were calculated and future analysis criteria for crime case work were estimated. Overall, the Precision ID Ancestry Panel performed well, and only minor changes to the recommended protocol were implemented. Three out of the 165 loci (rs459920, rs7251928, and rs7722456) had consistently poor performance, mainly due to misalignment of homopolymeric stretches. We suggest that these loci should be excluded from the analyses. The different statistical methods for reporting ancestry in forensic genetic case work are discussed.

Statistical modelling of Ion PGM HID STR 10-plex MPS data.

We investigated the results of short tandem repeat (STR) markers of dilution series experiments and reference profiles generated using the Ion PGM massively parallel sequencing platform utilising the HID STR 10-plex panel. The STR markers were identified by the marker specific flanking regions of the STR region. We investigated the following: (1) the usage of quality measures for identifying substitution errors, (2) the heterozygote balance and compared it to that of capillary electrophoresis (CE), (3) the stability of the coverage and the consequence of IonExpress Barcode adapter (IBA) sampling with decreasing amounts of template DNA, (4) the hypothesis that the parental longest uninterrupted stretch (LUS) is a better linear predictor of stutter ratio than the parent allele length, (5) the use of parental allele length as a predictor of shoulder ratio, and (6) the removal of non-systematic erroneous sequences using dynamic thresholds created by fitting the distribution of the non-systematic erroneous sequences. We found that, due to MID sampling, the average coverage on a marker could not be used as an apt predictor of the amount of template DNA. The parental LUS was shown to be better predictor of stutter ratio than the parental allele repeat length, when markers with compound and complex repeat patterns or markers which contained micro-variants were considered, such as marker TH01 showed R2 of 0.02 and 0.78 for parent allele repeat length and LUS, respectively. The one-inflated negative binomial method (OINB) and geometric model that can be used to remove non-systematic noise left on average 1.8 and 1.2 systematic errors per STR system, respectively.

Increasing the reference populations for the 55 AISNP panel: the need and benefits.

Ancestry inference for an individual can only be as good as the reference populations with allele frequency data on the SNPs being used. If the most relevant ancestral population(s) does not have data available for the SNPs studied, then analyses based on DNA evidence may indicate a quite distantly related population, albeit one among the more closely related of the existing reference populations. We have added reference population allele frequencies for 14 additional population samples (with >1100 individuals studied) to the 125 population samples previously published for the Kidd Lab 55 AISNP panel. Allele frequencies are now publicly available for all 55 SNPs in ALFRED and FROG-kb for a total of 139 population samples. This Kidd Lab panel of 55 ancestry informative SNPs has been incorporated in commercial kits by both ThermoFisher Scientific and Illumina for massively parallel sequencing. Researchers employing those kits will find the enhanced set of reference populations useful.

Frequencies of HID-ion ampliseq ancestry panel markers among greenlanders.

The HID-Ion AmpliSeq Ancestry Panel from Life Techologies includes 123 SNPs from the Seldin panel and 55 SNPs from Kidd panel in a single multiplex assay that helps to determine the continental biogeographic ancestry of individuals. We tested the panel on 104 Greenlanders, divided into a training set of 89 individuals and a test set of 15 individuals. All loci showed genotype distributions consistent with Hardy-Weinberg expectations. Linkage disequilibrium tests indicated that 14 pairs of loci were in association in Greenlanders. Population assignment of the training set to populations included in the HID SNP genotyper plugin placed most individuals in American, Asian, and in a few cases European populations. By including the genotype frequencies of this training set as a possible population of origin, all 15 individuals from the test set were correctly predicted to be Greenlanders using the Seldin SNPs, and nine were classified as Greenlanders using the Kidd SNPs. Population structure analysis indicated that Greenlanders have a genetic profile that is distinguishable from those of populations from America or Asia.

Effectiveness of structured, hospital-based, nurse-led atrial fibrillation clinics: a comparison between a real-world population and a clinical trial population.

OBJECTIVE: A previous randomised trial showed that structured, nurse-led atrial fibrillation (AF) care is superior to conventional AF care, although further research is needed to determine the outcomes of such care in a real-world setting. We compared the outcomes of patients in real-world, nurse-led, structured hospital AF clinics with the outcomes of a randomised trial of the efficacy of a nurse-led AF clinic, with respect to a composite outcome of cardiovascular-related hospitalisation and death. METHODS: All patients were referred to the AF nurse specialist by cardiologists. The AF nurse specialist provided patient education, risk-factor control and stimulated empowerment and compliance. During follow-up, treatment was adjusted according to clinical guidelines. Patient education was repeated, and compliance with medical treatment was controlled. The study size was powered as a non-inferiority study. Outcome measures were adjudicated by the same principles in both cohorts. RESULTS: A total of 596 patients from the real world and 356 patients from a clinical trial were included in this study. No significant difference between groups with respect to age, type of AF or CHA2DS2VASc score was found. The composite primary end point occurred with an incidence rate of 8.0 (95% CI 6.1 to 10.4) per 100 person-years in the real-world population and 8.3 (95% CI 6.3 to 10.9) per 100 person-years in the clinical trial, with a crude HR of 0.83 (95% CI 0.56 to 1.23). CONCLUSIONS: Structured, nurse-led, hospital-based AF care appears to be effective, and patient outcomes in an actual, hospital-based, structured AF care are as least as good as those in trial settings.

Levanase from Bacillus subtilis hydrolyses ß-2,6 fructosyl bonds in bacterial levans and in grass fructans.

A Levanase, LevB, from Bacillus subtilis 168, was expressed as a His6-tagged protein in Escherichia coli. The enzyme was purified and characterised for its activity and substrate specificity. LevB has a pH optimum of 6.0-6.5 and a maximum observed specific activity of 3 U mg(-1) using levan from Erwinia herbicola as substrate. Hydrolysis products were analysed by HPAEC, TLC, and NMR using chicory root inulin, mixed linkage fructans purified from ryegrass (Lolium perenne) and levan from E. herbicola as substrates. This revealed that LevB is an endolevanase that selectively cleaves the (ß-2,6) fructosyl bonds and does not hydrolyse inulin. Ryegrass fructans and bacterial levan was hydrolysed partially releasing oligosaccharides, but together with exoinulinase, LevB hydrolysed both ryegrass fructans and bacterial levan to near completion. We suggest that LevB can be used as a tool to achieve more structural information on complex fructans and to achieve complete degradation and quantification of mixed linkage fructans.