HaplotagLR: An efficient and configurable utility for haplotagging long reads.

Understanding the functional effects of sequence variation is crucial in genomics. Individual human genomes contain millions of variants that contribute to phenotypic variability and disease risks at the population level. Because variants rarely act in isolation, we must consider potential interactions of neighboring variants to accurately predict functional effects. We can accomplish this using haplotagging, which matches sequencing reads to their parental haplotypes using alleles observed at known heterozygous variants. However, few published tools for haplotagging exist and these share several technical and usability-related shortcomings that limit applicability, in particular a lack of insight or control over error rates, and lack of key metrics on the underlying sources of haplotagging error. Here we present HaplotagLR: a user-friendly tool that haplotags long sequencing reads based on a multinomial model and existing phased variant lists. HaplotagLR is user-configurable and includes a basic error model to control the empirical FDR in its output. We show that HaplotagLR outperforms the leading haplotagging method in simulated datasets, especially at high levels of specificity, and displays 7% greater sensitivity in haplotagging real data. HaplotagLR advances both the immediate utility of haplotagging and paves the way for further improvements to this important method.

LRphase: an efficient method for assigning haplotype identity to long reads.

Understanding the functional effects of sequence variation is among the primary goals of contemporary genomics. Individual human genomes contain millions of variants which are thought to contribute to phenotypic variability and differential disease risks at the population level. However, because variants rarely act in isolation, we cannot accurately predict functional effects without first considering the potential effects of other interacting variants on the same chromosome. This information can be obtained by phasing the read data from sequencing experiments. However, no standalone tools are available to simply phase reads based on known haplotypes. Here we present LRphase: a user-friendly utility for simple phasing of long sequencing reads.

The Inducible lac Operator-Repressor System Is Functional in Zebrafish Cells.

BACKGROUND: Zebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, an E. coli lac operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish. RESULTS: Here we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish. CONCLUSION: Our results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.

Pathogenicity in POLG syndromes: DNA polymerase gamma pathogenicity prediction server and database.

DNA polymerase gamma (POLG) is the replicative polymerase responsible for maintaining mitochondrial DNA (mtDNA). Disorders related to its functionality are a major cause of mitochondrial disease. The clinical spectrum of POLG syndromes includes Alpers-Huttenlocher syndrome (AHS), childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), the ataxia neuropathy spectrum (ANS) and progressive external ophthalmoplegia (PEO). We have collected all publicly available POLG-related patient data and analyzed it using our pathogenic clustering model to provide a new research and clinical tool in the form of an online server. The server evaluates the pathogenicity of both previously reported and novel mutations. There are currently 176 unique point mutations reported and found in mitochondrial patients in the gene encoding the catalytic subunit of POLG, POLG. The mutations are distributed nearly uniformly along the length of the primary amino acid sequence of the gene. Our analysis shows that most of the mutations are recessive, and that the reported dominant mutations cluster within the polymerase active site in the tertiary structure of the POLG enzyme. The POLG Pathogenicity Prediction Server (http://polg.bmb.msu.edu) is targeted at clinicians and scientists studying POLG disorders, and aims to provide the most current available information regarding the pathogenicity of POLG mutations.

Protein-altering and regulatory genetic variants near GATA4 implicated in bicuspid aortic valve.

Bicuspid aortic valve (BAV) is a heritable congenital heart defect and an important risk factor for valvulopathy and aortopathy. Here we report a genome-wide association scan of 466 BAV cases and 4,660 age, sex and ethnicity-matched controls with replication in up to 1,326 cases and 8,103 controls. We identify association with a noncoding variant 151 kb from the gene encoding the cardiac-specific transcription factor, GATA4, and near-significance for p.Ser377Gly in GATA4. GATA4 was interrupted by CRISPR-Cas9 in induced pluripotent stem cells from healthy donors. The disruption of GATA4 significantly impaired the transition from endothelial cells into mesenchymal cells, a critical step in heart valve development.

The N-terminal domain of the Drosophila mitochondrial replicative DNA helicase contains an iron-sulfur cluster and binds DNA.

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys(68), Cys(71), Cys(102), and Cys(105)) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.

Mapping 136 pathogenic mutations into functional modules in human DNA polymerase γ establishes predictive genotype-phenotype correlations for the complete spectrum of POLG syndromes.

We establish the genotype-phenotype correlations for the complete spectrum of POLG syndromes by refining our previously described protocol for mapping pathogenic mutations in the human POLG gene to functional clusters in the catalytic core of the mitochondrial replicase, Pol γ (1). We assigned 136 mutations to five clusters and identify segments of primary sequence that can be used to delimit the boundaries of each cluster. We report that compound heterozygotes with two mutations from different clusters manifested more severe, earlier-onset POLG syndromes, whereas two mutations from the same cluster are less common and generally are associated with less severe, later onset POLG syndromes. We also show that specific cluster combinations are more severe than others and have a higher likelihood to manifest at an earlier age. Our clustering method provides a powerful tool to predict the pathogenic potential and predicted disease phenotype of novel variants and mutations in POLG, the most common nuclear gene underlying mitochondrial disorders. We propose that such a prediction tool would be useful for routine diagnostics for mitochondrial disorders. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol γ.

Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. Recessive mutations in Pol γ represent nearly half of those reported to date, and they are nearly uniformly distributed along the length of the POLG1 gene (Human DNA Polymerase gamma Mutation Database); the majority of them are linked to the most severe form of POLG syndrome, Alpers-Huttenlocher syndrome. In this report, we assess the structure-function relationships for recessive disease mutations by reviewing existing biochemical data on site-directed mutagenesis of the human, Drosophila and yeast Pol γs, and their homologs from the family A DNA polymerase group. We do so in the context of a molecular model of Pol γ in complex with primer-template DNA, which we have developed based upon the recently solved crystal structure of the apoenzyme form. We present evidence that recessive mutations cluster within five distinct functional modules in the catalytic core of Pol γ. Our results suggest that cluster prediction can be used as a diagnosis-supporting tool to evaluate the pathogenic role of new Pol γ variants.

4-(4-Pyridylamino)pyridinium perchlorate.

In the title salt, C(10)H(10)N(3) (+)·ClO(4) (-), the 4-(4-pyridylamino)-pyridinium cations are linked into chains via N-H⋯N hydrogen bonding and into layers by C-H⋯π inter-actions [C⋯Cg = 3.3875 (19) Å]. Perchlorate ions are anchored to the layer motifs by N-H⋯O hydrogen bonding. The perchlorate anion was found to be disordered about a Cl-O axis, with two sites, each of equal occupancy, being resolved for the three remaining O atoms.

Bis(perchlorato-κO)tetra-kis[1-(2-pyridyl)-4-(4-pyridylmethyl-κN)piperazine]cadmium(II).

In the title compound, [Cd(ClO(4))(2)(C(15)H(18)N(4))(4)], the Cd(II) ion is coordinated in a slightly distorted octa-hedral environment by two trans monodentate perchlorate ligands and four 1-(2-pyrid-yl)-4-(4-pyridylmeth-yl)piperazine (pmpp) ligands. In the crystal structure, mol-ecules are organized into layers parallel to the ab plane by C-H⋯O inter-actions. Similar inter-actions promote the stacking of these layers into the three-dimensional crystal structure.

1,4-Bis(4-pyridylmeth-yl)piperazin-1-ium perchlorate fumaric acid hemisolvate.

In the title salt, C(16)H(21)N(4) (+)·ClO(4) (-)·0.5C(4)H(4)O(4), fumaric acid mol-ecules, situated across crystallographic inversion centres, are O-H⋯N hydrogen bonded to two protonated 1,4-bis-(4-pyridylmeth-yl)piperazine cations, forming trimolecular units. These construct one-dimensional supra-molecular ribbons by N-H⋯N hydrogen bonding, and further aggregate via π-π inter-actions [shortest C⋯C contact = 3.640 (1) Å] and perchlorate-mediated C-H⋯O inter-actions.

Poly[[diaqua-bis(µ(3)-maleato-κO:O,O:O)dicopper(II)] trihydrate].

In the title compound, {[Cu(2)(C(4)H(2)O(4))(2)(H(2)O)(2)]·3H(2)O}(n), Cu(II) ions with square-pyramidal coordination are bridged by exo-tri-dentate maleate dianions into [Cu(2)(maleate)(2)(H(2)O)(2)](n) layers coincident with the bc crystal plane. The inter-lamellar regions contain hydrogen-bonded cyclic water hexa-mers which facilitate layer stacking into a pseudo-three-dimensional crystal structure. The water hexamers themselves are formed by the operation of crystallographic inversion centers on sets of three crystallographically distinct water molecules of hydration.

catena-Poly[[[(dimethyl-malonato-κO:O')(perchlorato-κO)copper(II)]-µ-bis-(3-pyridylmeth-yl)piperazinediium-κN:N] perchlorate dihydrate].

In the title compound, {[Cu(C(5)H(6)O(4))(ClO(4))(C(16)H(22)N(4))]ClO(4)·2H(2)O}(n), square-pyramidally coordinated Cu atoms with perchlorate and dimethyl-malonate ligands are connected into cationic sinusoidal coordination polymer chains by doubly protonated bis-(3-pyridylmeth-yl)piperazine (3-bpmp) ligands. The chains aggregate into pseudo-layers parallel to the (101) crystal planes by N-H⋯O hydrogen bonding. Unligated perchlorate anions and water mol-ecules of crystallization provide additional hydrogen bonding between pseudo-layers.

Poly[4,4'-imino-dipyridinium [tetra-µ(3)-oxido-tetra-oxido-di-µ(4)-phosphato-κO:O':O'':O'''-tetra-vanadium(V)]].

In the title salt, {(C(10)H(11)N(3))[V(4)O(8)(PO(4))(2)]}(n), cubane-like [V(4)O(8)](4+) clusters are connected by phosphate anions into anionic [V(4)P(2)O(16)](n) (2n-) layers. These aggregate into the three-dimensional structure via N-H⋯O hydrogen-bonding mechanisms imparted by 4,4'-imino-dipyridinium dications situated between the layers.

Bis(dimethyl-malonato-κO,O')bis-[4-(4-pyridylamino-κN)pyridinium]nickel(II) hexa-hydrate.

In the title compound, [Ni(C(5)H(6)O(4))(2)(C(10)H(10)N(3))(2)]·6H(2)O, divalent nickel ions situated on the crystallographic twofold axis are octa-hedrally coordinated by four O atoms from two dimethyl-malonate ligands in a 1,3-chelating mode and two N atoms from two protonated monodentate 4,4'-dipyridylamine mol-ecules. The mol-ecules link into chains via N-H⋯O hydrogen bonding mediated by protonated pyridyl groups. The chains form layer patterns via π-π stacking [centroid-centroid distance = 3.777 (2) Å] . Water mol-ecule hexa-mers are generated from the unligated water mol-ecules (three per asymmetric unit) by inversion centers at Wyckoff position d. These clusters are situated between the pseudolayers, providing hydrogen-bonding pathways that build up the three-dimensional structure.