VarGenius executes cohort-level DNA-seq variant calling and annotation and allows to manage the resulting data through a PostgreSQL database.

BACKGROUND: Targeted resequencing has become the most used and cost-effective approach for identifying causative mutations of Mendelian diseases both for diagnostics and research purposes. Due to very rapid technological progress, NGS laboratories are expanding their capabilities to address the increasing number of analyses. Several open source tools are available to build a generic variant calling pipeline, but a tool able to simultaneously execute multiple analyses, organize, and categorize the samples is still missing. RESULTS: Here we describe VarGenius, a Linux based command line software able to execute customizable pipelines for the analysis of multiple targeted resequencing data using parallel computing. VarGenius provides a database to store the output of the analysis (calling quality statistics, variant annotations, internal allelic variant frequencies) and sample information (personal data, genotypes, phenotypes). VarGenius can also perform the "joint analysis" of hundreds of samples with a single command, drastically reducing the time for the configuration and execution of the analysis. VarGenius executes the standard pipeline of the Genome Analysis Tool-Kit (GATK) best practices (GBP) for germinal variant calling, annotates the variants using Annovar, and generates a user-friendly output displaying the results through a web page. VarGenius has been tested on a parallel computing cluster with 52 machines with 120GB of RAM each. Under this configuration, a 50 M whole exome sequencing (WES) analysis for a family was executed in about 7 h (trio or quartet); a joint analysis of 30 WES in about 24 h and the parallel analysis of 34 single samples from a 1 M panel in about 2 h. CONCLUSIONS: We developed VarGenius, a "master" tool that faces the increasing demand of heterogeneous NGS analyses and allows maximum flexibility for downstream analyses. It paves the way to a different kind of analysis, centered on cohorts rather than on singleton. Patient and variant information are stored into the database and any output file can be accessed programmatically. VarGenius can be used for routine analyses by biomedical researchers with basic Linux skills providing additional flexibility for computational biologists to develop their own algorithms for the comparison and analysis of data. The software is freely available at: https://github.com/frankMusacchia/VarGenius.

The nicotinic receptor beta 2 subunit is mutant in nocturnal frontal lobe epilepsy.

Clustered attacks of epileptic episodes originating from the frontal lobe during sleep are the main symptoms of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE, MIM 600513). Despite the clinical homogeneity, three forms of ADNFLE have been associated with chromosomes 20 (ENFL1; ref. 1), 15 (ENFL2; ref. 2) and 1 (ENFL3; ref. 3). Mutations of the gene encoding the neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4 ) have been found in ADNFLE-ENFL1 families, but these mutations account for only a small proportion of ADNFLE cases. The newly identified locus associated with ENFL3 harbours several candidate genes, including CHRNB2 (ref. 8), whose gene product, the beta 2 nicotinic acetylcholine receptor (nAChR) subunit, co-assembles with the alpha 4 nAChR subunit to form the active receptor.

Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat.

Fibronectin isoforms are generated by the alternative splicing of a primary transcript derived from a single gene. In rat at least three regions of the molecule are involved: EIIIA, EIIIB, and V. This study investigated the splicing patterns of these regions during development and aging, by means of ribonuclease protection analysis. Between fetal and adult rat, the extent of inclusion of the EIIIA and/or EIIIB region in fibronectin mRNA varied according to the type of tissue analyzed; but the inclusion of the V region, and in particular the V25 alternative variant, was significantly higher in all fetal than in adult tissues. These data suggest a crucial role of the V25 variant, possibly related to its interaction with the alpha 4 beta 1 integrin receptor during development. On the other hand, during aging, the only significant change observed in the splicing pattern was a decrease in the EIIIA variant in brain. The high inclusion levels of the EIIIA and EIIIB regions in young adult brain suggest that these segments may play an important role in differentiated brain tissue. The decreasing levels of inclusion of the EIIIA segment in brain fibronectin mRNA during aging may be an age-related marker with functional consequences.

Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption.

Epithelia permit selective and regulated flux from apical to basolateral surfaces by transcellular passage through cells or paracellular flux between cells. Tight junctions constitute the barrier to paracellular conductance; however, little is known about the specific molecules that mediate paracellular permeabilities. Renal magnesium ion (Mg2+) resorption occurs predominantly through a paracellular conductance in the thick ascending limb of Henle (TAL). Here, positional cloning has identified a human gene, paracellin-1 (PCLN-1), mutations in which cause renal Mg2+ wasting. PCLN-1 is located in tight junctions of the TAL and is related to the claudin family of tight junction proteins. These findings provide insight into Mg2+ homeostasis, demonstrate the role of a tight junction protein in human disease, and identify an essential component of a selective paracellular conductance.

Molecular basis of inherited spastic paraplegias.

Recently, paraplegin and spastin have been found to be mutated in two autosomal forms of hereditary spastic paraplegia. Both proteins harbour a common ATPase domain that expresses a chaperone function. Paraplegin is a nuclear-encoded mitochondrial metalloprotease, while the exact role and subcellular localisation of spastin are still unclear.

Familial hemiplegic migraine type 2 does not share hypersensitivity to nitric oxide with common types of migraine.

Familial hemiplegic migraine type 2 (FHM-2) and common types of migraine show phenotypic similarities which may indicate a common neurobiological background. The nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway plays a crucial role in migraine pathophysiology. Therefore, we tested the hypothesis that ATP1A2 mutations in patients with FHM-2 are associated with hypersensitivity to NO-cGMP pathway. Eight FHM-2 patients with R202Q, R763C, V138A and L764P mutations and nine healthy controls received intravenous infusions of 0.5 mug kg(-1) min(-1) glyceryl trinitrate (GTN) over 20 min. We recorded the following variables: headache intensity on a verbal rating scale; mean flow velocity in the middle cerebral artery (V(meanMCA)) by transcranial Doppler; diameter of the superficial temporal artery (STA) by ultrasound. The primary end-points were differences in incidence of migraine headache and area under the curve (AUC) for headache score during an immediate phase (0-120 min) and a delayed phase (2-14 h) after start of infusion. We found no difference in the incidence of reported migraine between FHM-2 patients, 25% (two out of eight), and controls, 0% (0 out of nine) (95% confidence interval -0.06, 0.56) (P = 0.21). The AUC(headache) in the immediate (P = 0.37) and delayed (P = 0.09) phase was not different between patients and controls. The GTN infusion resulted in a biphasic response in patients. During the immediate phase, the median peak headache occurred at 30 min and tended to be higher in patients, 1 (0, 3.8), than in controls, 0 (0, 1) (P = 0.056). During the delayed phase, the median peak headache occurred 4 h after the start of the infusion and was significantly higher in patients, 2.5 (0, 3), than in controls, 0 (0, 0) (P = 0.046). We found no difference in the AUC(VmeanMCA) (P = 0.77) or AUC(STA) (P = 0.53) between FHM-2 patients and controls. GTN infusion failed to induce more migraine in FHM-2 patients than in controls. The pathophysiological pathways underlying migraine headache in FHM-2 may be different from the common types of migraine.

A single site mutation in a truncated Fos protein allows it to interact with the TRE in vitro.

The Fos and Jun proteins, which are components of the transcription factor AP1, associate through the interaction of their so-called leucine zipper domains and bind strongly and specifically to DNA at phorbol ester-responsive elements. Jun also homodimerizes and binds the same element whereas Fos seems to have no specific affinity for DNA. We show that a single amino-acid change in the leucine zipper of Fos is sufficient to allow a truncated Fos protein to homodimerize and thus form a complex with DNA, even in the absence of Jun. This Fos-derived homodimer recognizes the consensus phorbol-ester responsive element specifically, in vitro. We conclude that the structural requirements for specific DNA binding are present in the Fos protein itself, with the exception of its lack of self-affinity.

Structure-derived hydrophobic potential. Hydrophobic potential derived from X-ray structures of globular proteins is able to identify native folds.

We present a model for the hydrophobic interaction in globular proteins that is based entirely on an analysis of known X-ray structures. This structure-derived hydrophobic force is identified as the strongest among the non-covalent interactions that stabilize native folds. The functional form of the hydrophobic interaction is found to be linear, corresponding to a constant force along the observable distance range (5 to 70 A). The parameters of the hydrophobic amino acid pair potentials yield a structure-derived hydrophobicity scale that correlates strongly with scales derived by a variety of complementary approaches. We demonstrate that the structure-derived hydrophobic interaction alone is able to distinguish a substantial number of native conformations from a large pool of misfolded structures.

Identification of native protein folds amongst a large number of incorrect models. The calculation of low energy conformations from potentials of mean force.

We present an approach that is able to detect native folds amongst a large number of non-native conformations. The method is based on the compilation of potentials of mean force of the interactions of the C beta atoms of all amino acid pairs from a database of known three-dimensional protein structures. These potentials are used to calculate the conformational energy of amino acid sequences in a number of different folds. For a substantial number of proteins we find that the conformational energy of the native state is lowest amongst the alternatives. Exceptions are proteins containing large prosthetic groups, Fe-S clusters or polypeptide chains that do not adopt globular folds. We discuss briefly potential applications in various fields of protein structural research.

Antisense transcription at the TRPM2 locus as a novel prognostic marker and therapeutic target in prostate cancer.

Overwhelming evidence indicates that cancer is a genetic disease caused by the accumulation of mutations in oncogenes and tumor suppressor genes. It is also increasingly apparent, however, that cancer depends not only on mutations in these coding genes but also on alterations in the large class of non-coding RNAs. Here, we report that one such long non-coding RNA, TRPM2-AS, an antisense transcript of TRPM2, which encodes an oxidative stress-activated ion channel, is overexpressed in prostate cancer (PCa). The high expression of TRPM2-AS and its related gene signature were found to be linked to poor clinical outcome, with the related gene signature working also independently of the patient's Gleason score. Mechanistically, TRPM2-AS knockdown led to PCa cell apoptosis, with a transcriptional profile that indicated an unbearable increase in cellular stress in the dying cells, which was coupled to cell cycle arrest, an increase in intracellular hydrogen peroxide and activation of the sense TRPM2 gene. Moreover, targets of existing drugs and treatments were found to be consistently associated with high TRPM2-AS levels in both targeted cells and patients, ultimately suggesting that the measurement of the expression levels of TRPM2-AS allows not only for the early identification of aggressive PCa tumors, but also identifies a subset of at-risk patients who would benefit from currently available, but mostly differently purposed, therapeutic agents.

Computational comparisons of model genomes.

Complete genomes from model organisms provide new challenges for computational molecular biology. Novel questions emerge from the genome data obtained from the functional prediction of thousands of gene products. In this review, we present some approaches to the computational comparison of genomes, based on sequence and text analysis, and comparisons of genome composition and gene order.

New protein functions in yeast chromosome VIII.

The analysis of the 269 open reading frames of yeast chromosome VIII by computational methods has yielded 24 new significant sequence similarities to proteins of known function. The resulting predicted functions include three particularly interesting cases of translation-associated proteins: peptidyl-tRNA hydrolase, a ribosome recycling factor homologue, and a protein similar to cytochrome b translational activator CBS2. The methodological limits of the meaningful transfer of functional information between distant homologues are discussed.

Association of the alpha-adducin locus with essential hypertension.

Previous studies on genetic rat hypertension have shown that polymorphism within the alpha-adducin gene may regulate blood pressure. Adducin is a cytoskeletal protein that may be involved in cellular signal transduction and interacts with other membrane-skeleton proteins that affect ion transport across the cell membrane. There is a high homology between rat and human adducin and pathophysiological similarities between the Milan hypertensive rat strain and a subgroup of patients with essential hypertension. Thus, we designed a case-control study to test the possible association between the alpha-adducin locus and hypertension. One hundred ninety primary hypertensive patients were compared with 126 control subjects. All subjects were white and unrelated. Four multiallelic markers surrounding the alpha-adducin locus located in 4p16.3 were selected: D4S125 and D4S95 mapping at 680 and 20 kb centromeric, and D4S43 and D4S228/E24 mapping at 660 and 2500 kb telomeric. Alleles for each marker were pooled into groups. Comparisons between control subjects and hypertensive patients were carried out by testing the allele-disease association relative to the marker genotype. The maximal association occurred for D4S95 (chi 2(1) 13.33), which maps closest to alpha-adducin. These data suggest that a polymorphism within the alpha-adducin gene may affect blood pressure in humans.

Linkage mapping of a new syndromic form of X-linked mental retardation, MRXS7, associated with obesity.

A new syndromic form of X-linked mental retardation associated to obesity, MRXS7, has been localised to Xp11.3-Xq23 in a large Pakistani family. The ten affected males show clinical manifestations of mental retardation, obesity and hypogonadism. The family was genotyped by a set of microsatellite markers spaced at approximately 10 cM intervals on the X chromosome. Linkage to five adjacent microsatellite markers, mapping in the pericentromeric area, was established and a maximum two-point lod score of 3.86 was reached at zero recombination with marker DXS1106. Reduced recombination events around the centromere prevented precise mapping of the gene.

Characterisation and chromosomal localisation of the rat alpha- and beta-adducin-encoding genes.

A polymorphism in the genes encoding alpha- and beta-adducin (ADD) was described as being associated with blood-pressure variation in a genetically hypertensive strain of rats (MHS). ADD is a cytoskeletal heterodimeric protein which may be involved in cellular signal transduction and interacts with other membrane skeleton proteins which affect ion transport across the cell membrane. The cDNA encoding the alpha subunit of rat ADD was isolated using PCR methods. The cDNA consists of about 3900 bp and encodes a protein of 735 amino acids (aa) which shows 91% aa identity with the human counterpart. In spleen and kidney, three alternative spliced exons were found by PCR amplification and confirmed by RNase protection analysis. 17 inbred rat strains were genotyped for the polymorphism in the alpha- and beta-ADD genes. Chromosomal localisation mapped rat alpha-ADD on chromosome 14 and rat beta-ADD on chromosome 4.

Transcription efficiency of human apolipoprotein A-I promoter varies with naturally occurring A to G transition.

In human, the gene coding for apolipoprotein A-I (apo A-I), a protein of the plasma lipid transport system, is expressed only in the liver and the intestine. A naturally occurring A to G substitution in the promoter at position -78 was shown to be associated with high density lipoproteins (HDL) in females. We have studied the effect of this mutation on promoter activity using various lengths of promoter sequences and the CAT reporter gene system. Transient expression studies after introduction of these constructs into Hep 3B cells revealed that in the region spanning -330 to +1 of the promoter an A to G substitution increases the activity approximately twofold. On the other hand, when further upstream region (-1469 to +397) is also included, the promoter activity seems comparable in both alleles. Our results show how minimal sequence variations can affect the in vitro analysis of promoter activity.

Early onset autosomal dominant dementia with ataxia, extrapyramidal features, and epilepsy.

OBJECTIVE: To perform a clinical and molecular study of a large autosomal dominant family with a complex neurologic syndrome that comprises early-onset dementia, extrapyramidal and cerebellar features, and epilepsy. BACKGROUND: Early-onset forms of dementia often are caused by genetic factors. Mutations of three different genes-amyloid precursor protein (APP), presenilin 1 (PS-1), presenilin 2 (PS-2)-have been found in early-onset autosomal dominant forms of AD, of the human microtubule associated-protein tau gene (MAPT) in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), of the BRI gene in familial British dementia, of the PI12 gene in familial encephalopathy with neuroserpin inclusion bodies. Linkage to chromosome 3 has been found in familial nonspecific dementia (FND) and linkage to chromosome 20 has been found in Huntington disease (HD)-like neurodegenerative disease. Dementia may be a feature of other neurodegenerative diseases such as HD, dentatorubro-pallidoluysian atrophy (DRPLA), diseases caused by mutations of the prion protein gene (PRNP), spinocerebellar ataxias (SCA), and familial parkinsonism. METHODS: A southern Italian family with autosomal dominant dementia-plus was observed. The family includes 57 individuals in 5 generations (14 affected, 7 personally observed). The authors performed linkage analysis to APP, PS-1, PS-2, FTDP-17, BRI, PI12, FND, HD-like, SCA4, SCA5, SCA10, SCA11, SCA13, PARK1, PARK2, PARK3 loci; direct mutation analysis of HD, DRPLA, SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA12, and PRNP genes; and sequencing of the PRNP open reading frame. RESULTS: Linkage to the examined loci was excluded. All of the direct mutation analyses were negative excluding mutations in the examined genes. CONCLUSIONS: This family has a peculiar phenotype and molecular analyses excluded genes known to cause hereditary dementias.

Paraplegin gene analysis in hereditary spastic paraparesis (HSP) pedigrees in northeast England.

OBJECTIVE: To identify the frequency and characterize the phenotype of paraplegin mutations in the hereditary spastic paraparesis (HSP) population in the northeast of England. BACKGROUND: HSP is a disorder that shows both clinical and genetic heterogeneity. To date, 13 loci have been associated with an HSP phenotype, with the causative gene having been identified in four of these. Two autosomal genes have been identified, paraplegin and spastin, and two X-linked genes have been identified, L1CAM (cell adhesion molecule) and proteolipid protein. METHODS: Thirty HSP pedigrees from the northeast of England were analyzed for mutation in each of the 17 exons of the paraplegin gene. RESULTS: A single family with a paraplegin mutation was identified in which the paraplegin mutation co-segregates with an HSP phenotype in an apparent dominant manner. The authors also describe frequent polymorphism in the paraplegin gene in both the HSP and control populations. CONCLUSION: Mutations in the paraplegin gene are not a common cause of HSP in the northeast of England. The phenotype of the paraplegin-related HSP family described had several striking features including amyotrophy, raised creatine kinase, sensorimotor peripheral neuropathy, and oxidative phosphorylation defect on muscle biopsy.

A new locus for autosomal dominant nocturnal frontal lobe epilepsy maps to chromosome 1.

BACKGROUND: Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is caused by mutations in the alpha4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) gene, mapping on chromosome 20q13.2. A second ADNFLE locus was mapped on chromosome 15q24. OBJECTIVE: To report a new third ADNFLE locus on chromosome 1 in a large Italian family. METHODS: The authors performed a clinical and genetic study in a large, three-generation ADNFLE family from southern Italy, including eight affected individuals and three obligate carriers. RESULTS: The age at onset of seizures was around 9 years of age and all affected individuals manifested nocturnal partial seizures of frontal lobe origin. Interictal awake and sleep EEG recordings showed no definite epileptiform abnormalities in most patients. Ictal video-EEG showed that the attacks were partial seizures with a frontal lobe semiology. Intellectual and neurologic examinations, and brain CT or MRI results were always normal. Carbamazepine was effective in all treated patients. Exclusion mapping of the known loci linked to ADNFLE-ENFL1, and ENFL2, on chromosomes 20q13.2 and 15q24-was performed on the pedigree before starting the genome-wide linkage analysis. The whole genome scan mapping allowed the identification of a new ADNFLE locus spanning the pericentromeric region of chromosome 1. CONCLUSIONS: The authors provided evidence for a third locus associated to autosomal dominant nocturnal frontal lobe epilepsy on chromosome 1. Among the known genes mapping within this critical region, the ss2 subunit of the nicotinic receptor (CHRNB2) represents the most obvious candidate.