MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, ...faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.
Coronavirus disease-2019 (COVID-19) triggers systemic infection with involvement of the respiratory tract. There are some patients developing haemostatic abnormalities during their infection with a ...considerably increased risk of death.
Patients (
= 85) with SARS-CoV-2 infection attending the University Medical Center, Mainz, from 3 March to 15 May 2020 were retrospectively included in this study. Data regarding demography, clinical features, treatment and laboratory parameters were analyzed. Twenty patients were excluded for assessment of disseminated intravascular coagulation (DIC) and thrombotic microangiopathy (TMA) due to lack of laboratory data.
COVID-19 patients (
= 65) were investigated, 19 with uncomplicated, 29 with complicated, and 17 with critical course; nine (13.8%) died. Seven patients showed overt DIC according to the ISTH criteria. The fibrinogen levels dropped significantly in these patients, although not below 100 mg/dl. Hallmarks of TMA, such as thrombocytopenia and microangiopathic haemolytic anaemia, were not detected in any of our COVID-19 patients. ADAMTS13 activity was mildly to moderately reduced in 4/22 patients, all having strongly elevated procalcitonin levels.
DIC occurred in 7/65 COVID-19 patients but fibrinogen and platelet consumption were compensated in almost all. ADAMTS13 assays excluded TTP and hallmarks of classic TMA were absent in all investigated patients. We hypothesize that the lacking erythrocyte fragmentation and only mild platelet consumption in severe COVID-19 are due to a microangiopathy predominantly localized to the alveolar microcirculation with a low blood pressure gradient.
Morbus Fabry is a hereditary metabolic disorder with low prevalence and late clinical manifestation. A defect in the α-galactosidase gene leads to lysosomal accumulation of the glycolipid ...globotriaosylceramide (Gb3). Gb3 may be used for monitoring of enzyme replacement therapy (ERT), but diagnostic sensitivity is limited. Recently, globotriaosylsphingosine (lysoGb3) was introduced as a promising new marker with significantly better sensitivity 23. For Fabry diagnosis, clinical studies and possible therapy monitoring, we established a fast and reliable LC–MS/MS assay for quantification of lysoGb3 in human plasma. Protein precipitation and glycolipid extraction from EDTA plasma was performed using acetone/methanol. Samples were analyzed by UPLC–MS/MS in MRM mode. In contrast to HPLC with fluorescence detection, the LC–MS/MS method requires no derivatization, less sample preparation and less instrument analysis time (<3min). As internal standard (ISTD), a glycine derivative of lysoGb3 was synthesized, and the product was purified by HPLC. ISTD properties such as polarity (affecting extraction and elution), ionization and fragmentation pathway were almost identical compared to the analyte. The new LC–MS/MS assay for the Fabry marker lysoGb3 shows good performance and allowed for better discrimination between Fabry patients and controls than Gb3.
Fabry disease is an X-chromosomally inherited lysosomal storage disorder leading to accumulation of glycosphingolipids, mainly globotriaosylceramide (ceramide-trihexoside, Gb3). Concentrations of Gb3 ...in plasma and urine have been used to diagnose Fabry disease and to monitor enzyme replacement therapy with recombinant alpha-galactosidase.
Gb3 was purified from plasma or urine by combined liquid extraction/protein precipitation and solid-phase extraction, and was detected by flow-injection analysis electrospray mass spectrometry (MS) using multi-reaction-monitoring. Calibration was performed via standard addition using C17-Gb3 as internal standard. The most abundant isoforms were monitored for calculation of total Gb3.
A MS-based assay for quantification of Gb3 in plasma and urine was established and validated. Intra- and inter-assay coefficient of variation (CV) of the method were <or=12%. However, at low concentrations the CV was 16%. The linear range covers roughly two orders of magnitude, down to 0.54 mg/L in plasma and 0.07 mg/L in urine. Careful adjustment of tuning parameters was necessary to obtain identical isoform intensities and quantitative results on different mass spectrometers. Gb3 concentrations in healthy controls were <4 mg/L in EDTA-plasma and <10 microg/mmol creatinine in urine. Significantly increased Gb3 concentrations were found in plasma and urine from male and female patients with Fabry disease.
An improved MS protocol for Gb3 quantification has been developed, validated, and shown to be suitable for diagnosis and monitoring of Fabry patients.
Background. Glycogen storage disease type III (GSDIII) is a rare metabolic disorder with autosomal recessive inheritance, caused by deficiency of the glycogen debranching enzyme. There is a high ...phenotypic variability due to different mutations in the AGL gene. Methods and Results. We describe a 2.3-year-old boy from a nonconsanguineous Romanian family, who presented with severe hepatomegaly with fibrosis, mild muscle weakness, cardiomyopathy, ketotic fasting hypoglycemia, increased transaminases, creatine phosphokinase, and combined hyperlipoproteinemia. GSD type IIIa was suspected. Accordingly, genomic DNA of the index patient was analyzed by next generation sequencing of the AGL gene. For confirmation of the two mutations found, genetic analysis of the parents and grandparents was also performed. The patient was compound heterozygous for the novel mutation c.3235C>T, p.Gln107 9 ⁎ (exon 24) and the known mutation c.1589C>G, p.Ser53 0 ⁎ (exon 12). c.3235 >T, p.Gln107 9 ⁎ was inherited from the father, who inherited it from his mother. c.1589C>G, p.Ser53 0 ⁎ was inherited from the mother, who inherited it from her father. Conclusion. We report the first genetically confirmed case of a Romanian patient with GSDIIIa. We detected a compound heterozygous genotype with a novel mutation, in the context of a severe hepatopathy and an early onset of cardiomyopathy.
Cerebral hypometabolism and abnormal levels of amyloid beta (Aβ), total (t-tau) and phosphorylated tau (ptau) proteins in cerebrospinal fluid (CSF) are established biomarkers of Alzheimer's disease ...(AD). We examined the agreement between these biomarkers in a single center study of patients with AD of severity extending over a wide range. Forty seven patients (MMSE 21.4 ± 3.6, range 13-28 points) with incipient and probable AD underwent positron emission tomography with 18F-fluorodeoxyglucose (FDG-PET) and lumbar puncture for CSF assays of Aβ1-42, p-tau181, and t-tau. All findings were classified as either positive or negative for AD. Statistical analyses were performed for the whole sample (n=47) and for the subgroups stratified as mild (MMSE > 20 points, n=30) and moderate (MMSE < 21 points, n=17) AD. In the whole patient sample, the agreement with the FDG-PET finding was 77% (chance-corrected kappa κ=0.34, p=0.016) for t-tau, 68% (κ=0.10, n.s.) for p-tau181, and 68% (κ=0.04, n.s.) for Aβ1-42. No significant agreement was found in the mild AD subgroup, while there was a strong agreement for t-tau (94%, κ=0.77, p=0.001) and p-tau181 (88%, κ=0.60, p=0.014) in the moderate AD group. A significant agreement between the FDG-PET and CSF tau findings in patients with AD supports the view that both are markers of neurodegeneration. CSF tau proteins and FDG-PET might substitute each other as supportive diagnostic tools in patients with suspected moderate-to-severe Alzheimer's dementia, while this is not the case in subjects at an earlier disease stage.
Reliable PCR amplification of DNA fragments is the prerequisite for most genetic assays. We investigated the impact of G-quadruplex- or i-motif-like sequences on the reliability of PCR-based genetic ...analyses.
We found the sequence context of a common intronic polymorphism in the MEN1 gene (multiple endocrine neoplasia I) to be the cause of systematic genotyping errors by inducing preferential amplification of one allelic variant allele dropout (ADO). Bioinformatic analyses and pyrosequencing-based allele quantification enabled the identification of the underlying DNA structures.
We showed that G-quadruplex- or i-motif-like sequences can reproducibly cause ADO. In these cases, amplification efficiency strongly depends on the PCR enzyme and buffer conditions, the magnesium concentration in particular. In a randomly chosen subset of candidate single-nucleotide polymorphisms (SNPs) defined by properties deduced from 2 originally identified ADO cases, we confirmed preferential PCR amplification in up to 50% of the SNPs. We subsequently identified G-quadruplex and i-motifs harboring a SNP that alters the typical motif as the cause of this phenomenon, and a genomewide search based on the respective motifs predicted 0.5% of all SNPs listed by dbSNP and Online Mendelian Inheritance in Man to be potentially affected.
Undetected, the described phenomenon produces systematic errors in genetic analyses that may lead to misdiagnoses in clinical settings. PCR products should be checked for G-quadruplex and i-motifs to avoid the formation of ADO-causing secondary structures. Truly affected assays can then be identified by a simple experimental procedure, which simultaneously provides the solution to the problem.
The prognostic value of copeptin, the C-terminal fragment of the precursor protein of vasopressin which is released upon stress, and hypotension in pulmonary embolism is unknown, especially if ...combined with biomarkers reflecting different pathophysiological axes such as myocardial injury (high-sensitivity troponin T (hsTnT)) and stretch (N-terminal pro-brain natriuretic peptide (NT-proBNP)).We prospectively studied 268 normotensive pulmonary embolism patients included in a single-centre cohort study.Patients with an adverse 30-day outcome (5.6%) had higher copeptin levels than patients with a favourable course (median (interquartile range) 51.8 (21.6-90.8) versus 13.2 (5.9-39.3) pmol·L(-1); p=0.020). Patients with copeptin levels above the calculated optimal cut-off value of 24 pmol·L(-1) had a 5.4-fold increased risk for an adverse outcome (95% CI 1.68-17.58; p=0.005). We developed a strategy for risk stratification based on biomarkers. None of 141 patients (52.6%) with hsTnT <14 pg·mL(-1) or NT-proBNP <600 pg·mL(-1) had an adverse outcome (low risk). Copeptin ≥24 pmol·L(-1) stratified patients with elevated hsTnT and NT-proBNP as intermediate-low and intermediate-high risk (5.6% and 20.0% adverse outcome, respectively). Compared to the algorithm proposed by the 2014 European Society of Cardiology guideline, more patients were classified as low risk (52.8% versus 17.5%, p<0.001) and more patients in the intermediate-high risk group had an adverse outcome (20.0% versus 11.6%).Copeptin might be helpful for risk stratification of normotensive patients with pulmonary embolism, especially if integrated into a biomarker-based algorithm.
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