GPD1L-A306del modifies sodium current in a family carrying the dysfunctional SCN5A-G1661R mutation associated with Brugada syndrome.

Loss-of-function variants of SCN5A, encoding the sodium channel alpha subunit Nav1.5 are associated with high phenotypic variability and multiple cardiac presentations, while underlying mechanisms are incompletely understood. Here we investigated a family with individuals affected by Brugada Syndrome (BrS) of different severity and aimed to unravel the underlying genetic and electrophysiological basis.Next-generation sequencing was used to identify the genetic variants carried by family members. The index patient, who was severely affected by arrhythmogenic BrS, carried previously uncharacterized variants of Nav1.5 (SCN5A-G1661R) and glycerol-3-phosphate dehydrogenase-1-like protein (GPD1L-A306del) in a double heterozygous conformation. Family members exclusively carrying SCN5A-G1661R showed asymptomatic Brugada ECG patterns, while another patient solely carrying GPD1L-A306del lacked any clinical phenotype.To assess functional mechanisms, Nav1.5 channels were transiently expressed in HEK-293 cells in the presence and absence of GPD1L. Whole-cell patch-clamp recordings revealed loss of sodium currents after homozygous expression of SCN5A-G1661R, and reduction of current amplitude to ~ 50% in cells transfected with equal amounts of wildtype and mutant Nav1.5. Co-expression of wildtype Nav1.5 and GPD1L showed a trend towards increased sodium current amplitudes and a hyperpolarizing shift in steady-state activation and -inactivation compared to sole SCN5A expression. Application of the GPD1L-A306del variant shifted steady-state activation to more hyperpolarized and inactivation to more depolarized potentials.In conclusion, SCN5A-G1661R produces dysfunctional channels and associates with BrS. SCN5A mediated currents are modulated by co-expression of GDP1L and this interaction is altered by mutations in both proteins. Thus, additive genetic burden may aggravate disease severity, explaining higher arrhythmogenicity in double mutation carriers.

New cases that expand the genotypic and phenotypic spectrum of Congenital NAD Deficiency Disorder.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in over 400 cellular reactions. During embryogenesis, mammals synthesize NAD de novo from dietary l -tryptophan via the kynurenine pathway. Biallelic, inactivating variants in three genes encoding enzymes of this biosynthesis pathway (KYNU, HAAO, and NADSYN1) disrupt NAD synthesis and have been identified in patients with multiple malformations of the heart, kidney, vertebrae, and limbs; these patients have Congenital NAD Deficiency Disorder HAAO and four families with biallelic variants in KYNU. These patients present similarly with multiple malformations of the heart, kidney, vertebrae, and limbs, of variable severity. We show that each variant identified in these patients results in loss-of-function, revealed by a significant reduction in NAD levels via yeast genetic complementation assays. For the first time, missense mutations are identified as a cause of malformation and shown to disrupt enzyme function. These missense and frameshift variants cause moderate to severe NAD deficiency in yeast, analogous to insufficient synthesized NAD in patients. We hereby expand the genotypic and corresponding phenotypic spectrum of Congenital NAD Deficiency Disorder.

Prognostic significance of clinical, histopathological, and molecular characteristics of medulloblastomas in the prospective HIT2000 multicenter clinical trial cohort.

This study aimed to prospectively evaluate clinical, histopathological and molecular variables for outcome prediction in medulloblastoma patients. Patients from the HIT2000 cooperative clinical trial were prospectively enrolled based on the availability of sufficient tumor material and complete clinical information. This revealed a cohort of 184 patients (median age 7.6 years), which was randomly split at a 2:1 ratio into a training (n = 127), and a test (n = 57) dataset in order to build and test a risk score for this population. Independent validation was performed in a non-overlapping cohort (n = 83). All samples were subjected to thorough histopathological investigation, CTNNB1 mutation analysis, quantitative PCR, MLPA and FISH analyses for cytogenetic variables, and methylome analysis. By univariable analysis, clinical factors (M-stage), histopathological variables (large cell component, endothelial proliferation, synaptophysin pattern), and molecular features (chromosome 6q status, MYC amplification, subgrouping) were found to be prognostic. Molecular consensus subgrouping (WNT, SHH, Group 3, Group 4) was validated as an independent feature to stratify patients into different risk groups. When comparing methods for the identification of WNT-driven medulloblastoma, this study identified CTNNB1 sequencing and methylation profiling to most reliably identify these patients. After removing patients with particularly favorable (CTNNB1 mutation, extensive nodularity) or unfavorable (MYC amplification) markers, a risk score for the remaining "intermediate molecular risk" population dependent on age, M-stage, pattern of synaptophysin expression, and MYCN copy-number status was identified, with speckled synaptophysin expression indicating worse outcome. Test and independent validation of the score confirmed significant discrimination of patients by risk profile. Methylation subgrouping and CTNNB1 mutation status represent robust tools for the risk stratification of medulloblastoma. A simple clinico-pathological risk score was identified, which was confirmed in a test set and by independent clinical validation.

A novel frameshift mutation and infrequent clinical findings in two cases with Dyggve-Melchior-Clausen syndrome.

Dyggve-Melchior-Clausen syndrome (DMC) (MIM #223800) is a rare autosomal-recessive type of skeletal dysplasia accompanied by variable degrees of intellectual disability (ID). It is characterized by progressive spondyloepimetaphyseal dysplasia leading to disproportionate short stature, microcephaly, and coarse facies. The radiographic appearance of generalized platyspondyly with double-humped end plates and the lace-like appearance of iliac crests are pathognomonic in this syndrome. The disorder results from mutations in the dymeclin (DYM) mapped to the 18q12-12.1 chromosomal region. Here, we report two cases with DMC: one with disproportionate short stature, developmental delay, and severe ID with a novel frameshift mutation (c.1028_1056del29) leading to a premature stop codon, and the second patient with classical clinical and radiological features of DMC with mild ID and rectal prolapse, which is very rare. The clinical diagnosis was confirmed with molecular analysis of DYM with a known mutation at c.580C>T (p.R194X). The parents and sibling of the second patient were heterozygous carriers with mild skeletal changes and short stature.

Detection of Aspergillus DNA by a nested PCR assay is superior to blood culture in an experimental murine model of invasive aspergillosis.

For diagnosing invasive aspergillosis (IA), an increasing clinical problem in immunocompromised patients, molecular tools are gaining in importance. Detection of Aspergillus DNA in blood samples was investigated by a nested PCR assay in a murine model of experimentally induced IA. Ex vivo, the detection threshold of the PCR assay was determined in blood and organ homogenates of mice. After intravenous injection of Aspergillus fumigatus conidia on different days, growth of colonies was determined in cultures of blood and organs from immunocompetent and immunosuppressed mice and Aspergillus DNA was detected from blood samples by a nested PCR assay. The detection threshold of the PCR assay was as low as 1 c.f.u. ml(-1). The assay proved to be more sensitive than cultures of blood, with sensitivity rates between 17.6 and 87.5 % depending on the fungal burden. In conclusion, the nested PCR assay is superior to cultural methods in detecting Aspergillus spp. in murine blood samples.

Development of a LightCycler PCR assay for detection and quantification of Aspergillus fumigatus DNA in clinical samples from neutropenic patients.

The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCycler-based real-time PCR assay to detect and quantify rapidly, specifically, and sensitively Aspergillus fumigatus DNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from an A. fumigatus-specific sequence of the mitochondrial cytochrome b gene. The assay is linear in the range between 13.2 fg and 1.3 ng of A. fumigatus DNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples.

Clinical evaluation of a polymerase chain reaction assay to detect Aspergillus species in bronchoalveolar lavage samples of neutropenic patients.

The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. Using a recently developed two-step polymerase chain reaction (PCR) assay to detect 10 fg of Aspergillus DNA, corresponding to 1-5 colony-forming units (CFU)/ml of spiked samples in vitro, we prospectively examined 197 bronchoalveolar lavage (BAL) samples from 176 subjects, including 141 neutropenic, febrile patients with lung infiltrates, at risk for invasive fungal disease. Underlying diseases of these patients were haematological malignancies; 93 patients suffered from acute leukaemias. Thirty-one of these immunocompromised patients (17.6%) were PCR positive, correlating with positive BAL culture, positive histology from lung surgery or from autopsy, positive computerized tomography scans or positive galactomannan enzyme-linked immunosorbent assay. Six patients (4.3%) of this group had positive PCR results without any correlation to clinical or other diagnostic data, probably owing to contamination of the samples by ubiquitous Aspergillus spores. The samples of two patients (1.4%) with a subsequent histologically proven mould infection were PCR negative. All 102 immunocompromised patients (72.3%) with a negative PCR showed no evidence of invasive fungal disease. From 35 patients without immunodeficiency, four (11.4%) showed positive results, without evidence of invasive or non-invasive pulmonary aspergillosis. In this haematological population, the sensitivity and specificity values of the test reached 93.9% and 94.4%, the positive predictive value 83.8%, the negative predictive value 98.1%. Our data support the considerable clinical value of this PCR assay for confirming and improving diagnosis of pulmonary aspergillosis in high-risk patients.

SigB, an alternative sigma factor of the myxobacterium Stigmatella aurantiaca, is synthesized during development and heat shock.

Alternative sigma factors have been detected in the myxobacterium Stigmatella aurantiaca during indole-induced sporulation, fruiting body formation and heat shock using an antiserum raised against sigma factor SigB. The time course of sigB gene expression was analysed by RT-PCR and by determining beta-galactosidase activity during development in a merodiploid strain that harboured a sigB-lacZ fusion gene. Inactivation of the sigB gene by insertion of the neo gene resulted in the loss of one sigma factor as shown by Western analysis. Neither fruiting body formation nor sporulation, nor the production of possible SigB targets, such as DnaK, GroEL or HspA, were affected.