[Current modalities and concepts on access and use of biospecimen samples and associated data for research from human biobanks]. / Nutzungskonzepte für Proben und Daten aus humanen Biobanken für die Forschung.

It is accepted worldwide that biospecimen and data sharing (BDS) play an essential role for the future of medical research to improve diagnostics and prognostics, e.g. by validated biomarkers. BDS is also pivotal to the development of new therapeutic treatments and for the improvement of population health. Human biobanks can generate an added value to this need by providing biospecimens and/or associated data to researchers. An inspection of several examples of epidemiological as well as clinical/disease-oriented biobanks in Germany shows that best practice procedures (BPP) that are internationally agreed on are being installed for biospecimen and/or data access. In general, fair access is aimed at requiring a written application by the requesting scientist, which is then peer-reviewed for scientific and ethical validity by the Biobank. Applied BPP take into account (i) patient education/agreement according to the informed consent model, (ii) privacy protection, (iii) intellectual property rights, the (iv) notification obligation of health-related findings (including incidental findings), the (v) use of material (MTA) and data transfer agreements (DTA) for mutual legal security, the avoidance of conflicts of interests, as well as for cost recovery/fee for service as a basis for sustainability of the biobank. BPP are rooted in the self-regulation efforts of life sciences and are supported by parent ethics committees in Germany. Central biobank registries displaying aggregated information on biospecimens stored and the research foci constitute an important tool to make biobanks that are scattered across the country visible to each other, and, can thus promote access to hitherto unknown biospecimen and data resources.

X chromosomal variation is associated with slow progression to AIDS in HIV-1-infected women.

AIDS has changed from a mostly male-specific health problem to one that predominantly affects females. Although sex differences in HIV-1 susceptibility are beyond doubt, the extent to which sex affects the onset and progression of AIDS has remained elusive. Here, we provide evidence for an influence of X chromosomal variation on the course of retroviral infection, both in HIV-1-infected patients and in the rhesus macaque model of AIDS. A two-stage, microsatellite-based GWAS of SIV-infected monkeys revealed MHC class I markers and a hitherto-unknown X chromosomal locus as being associated with a nominal score measuring progression to AIDS (Fisher's exact p < 10(-6)). The X chromosomal association was subsequently confirmed in HIV-1-infected patients with published SNP genotype data. SNP rs5968255, located at human Xq21.1 in a conserved sequence element near the RPS6KA6 and CYLC1 genes, was identified as a significant genetic determinant of disease progression in females (ANOVA p = 8.8 x 10(-5)), but not in males (p = 0.19). Heterozygous female carriers of the C allele showed significantly slower CD4 cell decline and a lower viral load at set point than TT homozygous females and than males. Inspection of HapMap revealed that the CT genotype is significantly more frequent among Asians than among Europeans or Africans. Our results suggest that, in addition to the individual innate and adaptive immunity status, sex-linked genetic variation impacts upon the rate of progression to AIDS. Elucidating the mechanisms underlying this sex-specific effect will promote the development of antiretroviral therapies with high efficacy in both sexes.

Towards a Core Set of Indicators for Data Quality of Registries.

Registries are a widely accepted method in health services research. Registry owners are faced with the challenge to document and assure data quality, vital for answering research questions and conducting quality research. Therefore a survey on indicators for data quality was conducted as part of a German funding initiative. A list of 51 pre-defined quality indicators was provided to 16 patient registry projects in a web based survey. The assessment included three criteria derived from the Rand Appropriateness Method (RAM), the application area, and three criteria representing a project-specific perspective. Considering the criteria adapted from RAM, a core set of 17 indicators could be identified. This core set covered important dimensions, such as case completeness, data completeness and validity. Adding importance as a criterion from a project-specific perspective led to a subset of six indicators. The selection of indicators identified through this survey may be applied on different use cases, e.g. a) benchmarking between registries, b) benchmarking of study sites, and c) value-based remuneration of study sites. Thus, the presented core set of indicators can be used as a basis to improve quality of registry data with a systematic approach.

VarWatch-A stand-alone software tool for variant matching.

Massively parallel DNA sequencing of clinical samples holds great promise for the gene-based diagnosis of human inherited diseases because it allows rapid detection of putatively causative mutations at genome-wide level. Without additional evidence complementing their initial bioinformatics evaluation, however, the clinical relevance of such candidate genetic variants often remains unclear. In consequence, dedicated 'matching' services have been established in recent years that aim at the discovery of other, comparable case reports to facilitate individual diagnoses. However, legal concerns have been raised about the global sharing of genetic data, particularly in Europe where the recently enacted General Data Protection Regulation EU-2016/679 classifies genetic data as highly sensitive. Hence, unrestricted sharing of genetic data from clinical cases on platforms outside the national jurisdiction increasingly may be perceived as problematic. To allow collaborative data producers, particularly large consortia of diagnostic laboratories, to acknowledge these concerns while still practicing efficient case matching internally, novel tools are required. To this end, we developed VarWatch, an easy-to-deploy and highly scalable case matching software that provides users with comprehensive programmatic tools and a user-friendly interface to fulfil said purpose.

IT Infrastructure for Registries in Health Services Research: A Market Study in Germany.

Registries are increasingly implemented to record the practice of health care. Within a national funding scheme for registries, an accompanying project was launched to support the design of the registries' IT infrastructure amongst other tasks for 16 projects. A challenge of data management systems was organized by the accompanying project in order to enable the projects to define realistic expectations towards IT support in their research protocols. Twelve vendors participated in the challenge. They presented their solutions for selected use cases. In advance, the projects considered a sufficient authorization concept and the possibility to export data to be of highest importance. However, the systems covered mainly core processes of electronic data capture. The accompanying project will continue its support for the next stage of the funding scheme, which will be the implementation of the registries that win a competitive review of their research protocols prepared in the concept development stage.

The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli.

Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.

Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence.

BACKGROUND: Defensins are important components of innate immunity to combat bacterial and viral infections, and can even elicit antitumor responses. Clusters of defensin (DEF) genes are located in a 2 Mb range of the human chromosome 8p23.1. This DEF locus, however, represents one of the regions in the euchromatic part of the final human genome sequence which contains segmental duplications, and recalcitrant gaps indicating high structural dynamics. RESULTS: We find that inter- and intraindividual genetic variations within this locus prevent a correct automatic assembly of the human reference genome (NCBI Build 34) which currently even contains misassemblies. Manual clone-by-clone alignment and gene annotation as well as repeat and SNP/haplotype analyses result in an alternative alignment significantly improving the DEF locus representation. Our assembly better reflects the experimentally verified variability of DEF gene and DEF cluster copy numbers. It contains an additional DEF cluster which we propose to reside between two already known clusters. Furthermore, manual annotation revealed a novel DEF gene and several pseudogenes expanding the hitherto known DEF repertoire. Analyses of BAC and working draft sequences of the chimpanzee indicates that its DEF region is also complex as in humans and DEF genes and a cluster are multiplied. Comparative analysis of human and chimpanzee DEF genes identified differences affecting the protein structure. Whether this might contribute to differences in disease susceptibility between man and ape remains to be solved. For the determination of individual DEF gene repertoires we provide a molecular approach based on DEF haplotypes. CONCLUSIONS: Complexity and variability seem to be essential genomic features of the human DEF locus at 8p23.1 and provides an ongoing challenge for the best possible representation in the human reference sequence. Dissection of paralogous sequence variations, duplicon SNPs ans multisite variations as well as haplotypes by sequencing based methods is the way for future studies of interindividual DEF locus variability and its disease association.

Association of TLR7 variants with AIDS-like disease and AIDS vaccine efficacy in rhesus macaques.

In HIV infection, TLR7-triggered IFN-α production exerts a direct antiviral effect through the inhibition of viral replication, but may also be involved in immune pathogenesis leading to AIDS. TLR7 could also be an important mediator of vaccine efficacy. In this study, we analyzed polymorphisms in the X-linked TLR7 gene in the rhesus macaque model of AIDS. Upon resequencing of the TLR7 gene in 36 rhesus macaques of Indian origin, 12 polymorphic sites were detected. Next, we identified three tightly linked single nucleotide polymorphisms (SNP) as being associated with survival time. Genotyping of 119 untreated, simian immunodeficiency virus (SIV)-infected male rhesus macaques, including an 'MHC adjusted' subset, revealed that the three TLR7 SNPs are also significantly associated with set-point viral load. Surprisingly, this effect was not observed in 72 immunized SIV-infected male monkeys. We hypothesize (i) that SNP c.13G>A in the leader peptide is causative for the observed genotype-phenotype association and that (ii) the underlying mechanism is related to RNA secondary structure formation. Therefore, we investigated a fourth SNP (c.-17C>T), located 17 bp upstream of the ATG translation initiation codon, that is also potentially capable of influencing RNA structure. In c.13A carriers, neither set-point viral load nor survival time were related to the c.-17C>T genotype. In c.13G carriers, by contrast, the c.-17C allele was significantly associated with prolonged survival. Again, no such association was detected among immunized SIV-infected macaques. Our results highlight the dual role of TLR7 in immunodeficiency virus infection and vaccination and imply that it may be important to control human AIDS vaccine trials, not only for MHC genotype, but also for TLR7 genotype.

Theoretical study of lipid biosynthesis in wild-type Escherichia coli and in a protoplast-type L-form using elementary flux mode analysis.

In the present study, we investigated lipid biosynthesis in the bacterium Escherichia coli by mathematical modeling. In particular, we studied the interaction between the subsystems producing unsaturated and saturated fatty acids, phospholipids, lipid A, and cardiolipin. The present analysis was carried out both for the wild-type and for several in silico knockout mutants, using the concept of elementary flux modes. Our results confirm that, in the wild type, there are four main products: L1-phosphatidylethanolamine, lipid A, lipid A (cold-adapted), and cardiolipin. We found that each of these compounds is produced on several different routes, indicating a high redundancy of the system under study. By analysis of the elementary flux modes remaining after the knockout of genes of lipid biosynthesis, and comparison with publicly available data on single-gene knockouts in vivo, we were able to determine the metabolites essential for the survival of the cell. Furthermore, we analyzed a set of mutations that occur in a cell wall-free mutant of Escherichia coli W1655F+. We postulate that the mutant is not capable of producing both forms of lipid A, when the combination of mutations is considered to make a nonfunctional pathway. This is in contrast to gene essentiality data showing that lipid A synthesis is indispensable for the survival of the cell. The loss of the outer membrane in the cell wall-free mutant, however, shows that lipid A is dispensable as the main component of the outer surface structure in this particular E. coli strain.

GenColors: accelerated comparative analysis and annotation of prokaryotic genomes at various stages of completeness.

SUMMARY: GenColors is a new web-based software/database system aimed at an improved and accelerated annotation of prokaryotic genomes, considering information on related genomes and making extensive use of genome comparison. It offers a seamless integration of data from ongoing sequencing projects and annotated genomic sequences obtained from GenBank. The genome comparison tools determine, for example, best-bidirectional hits, gene conservation, syntenies and gene core sets. Swiss-Prot/TrEMBL hits allow annotations in an effective manner. To further support the annotation base-specific quality data can also be displayed if available. With GenColors dedicated genome browsers containing a group of related genomes can be easily set up and maintained. It has been efficiently used for Borrelia garinii and is currently applied to various ongoing genome projects. AVAILABILITY: Detailed information on GenColors is available at http://gencolors.imb-jena.de. Online usage of GenColors-based genome browsers is the preferred application mode. The system is also available upon request for local installation.

Mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from Ralstonia eutropha.

Molybdenum enzymes containing the pterin cofactor are a diverse group of enzymes that catalyse in general oxygen atom transfer reactions. Aiming at studying the amino acid residues, which are important for the enzymatic specificity, we used nitrate reductase from Ralstonia eutropha (R.e.NAP) as a model system for mutational studies at the active site. We mutated amino acids at the Mo active site (Cys181 and Arg421) as well as amino acids in the funnel leading to it (Met182, Asp196, Glu197, and the double mutant Glu197-Asp196). The mutations were made on the basis of the structural comparison of nitrate reductases with formate dehydrogenases (FDH), which show very similar three-dimensional structures, but clear differences in amino acids surrounding the active site. For mutations Arg421Lys and Glu197Ala we found a reduced nitrate activity while the other mutations resulted in complete loss of activity. In spite of the partial of total loss of nitrate reductase activity, these mutants do not, however, display FDH activity.

Mutagenesis study on the role of a lysine residue highly conserved in formate dehydrogenases and periplasmic nitrate reductases.

Lysine 85 (K85) in the primary structure of the catalytic subunit of the periplasmic nitrate reductase (NAP-A) of Ralstonia eutropha H16 is highly conserved in periplasmic nitrate reductases and in the structurally related catalytic subunit of the formate dehydrogenases of various bacterial species. It is located between an [4Fe-4S] center and one of the molybdopterin-guanine dinucleotides mediating the through bonds electron flow to convert the specific substrate of the respective enzymes. To examine the role of K85, the structure of NAP-A of R. eutropha strain H16 was modeled on the basis of the crystal structure from the Desulfovibrio desulfuricans enzyme (Dias et al. Structure Fold Des. 7(1) (1999) 65) and K85 was replaced by site-directed mutagenesis, yielding K85R and K85M, respectively. The specific nitrate reductase activity was determined in periplasmic extracts. The mutant enzyme carrying K85R showed 23% of the wild-type activity, whereas the replacement by a polar, uncharged residue (K85M) resulted in complete loss of the catalytic activity. The reduced nitrate reductase activity of K85R was not due to different quantities of the expressed gene product, as controlled immunologically by NAP-specific antibodies. The results indicate that K85 is optimized for the electron transport flux to reduce nitrate to nitrite in NAP-A, and that the positive charge alone cannot meet further structural requirement for efficient electron flow.