Changing genetic paradigms: creating next-generation genetic databases as tools to understand the emerging complexities of genotype/phenotype relationships

Understanding genotype/phenotype relationships has become more complicated as increasing amounts of inter- and intra-tissue genetic heterogeneity have been revealed through next-generation sequencing and evidence showing that factors such as epigenetic modifications, non-coding RNAs and RNA editing can play an important role in determining phenotype. Such findings have challenged a number of classic genetic assumptions including (i) analysis of genomic sequence obtained from blood is an accurate reflection of the genotype responsible for phenotype expression in an individual; (ii) that significant genetic alterations will be found only in diseased individuals, in germline tissues in inherited diseases, or in specific diseased tissues in somatic diseases such as cancer; and (iii) that mutation rates in putative disease-associated genes solely determine disease phenotypes. With the breakdown of our traditional understanding of genotype to phenotype relationships, it is becoming increasingly apparent that new analytical tools will be required to determine the relationship between genotype and phenotypic expression. To this end, we are proposing that next-generation genetic database (NGDB) platforms be created that include new bioinformatics tools based on algorithms that can evaluate genetic heterogeneity, as well as powerful systems biology analysis tools to actively process and evaluate the vast amounts of both genomic and genomic-modifying information required to reveal the true relationships between genotype and phenotype.     

Going the Distance for Protein Function Prediction: A New Distance Metric for Protein Interaction Networks

In protein-protein interaction (PPI) networks, functional similarity is often inferred based on the function of directly interacting proteins, or more generally, some notion of interaction network proximity among proteins in a local neighborhood. Prior methods typically measure proximity as the shortest-path distance in the network, but this has only a limited ability to capture fine-grained neighborhood distinctions, because most proteins are close to each other, and there are many ties in proximity. We introduce diffusion state distance (DSD), a new metric based on a graph diffusion property, designed to capture finer-grained distinctions in proximity for transfer of functional annotation in PPI networks. We present a tool that, when input a PPI network, will output the DSD distances between every pair of proteins. We show that replacing the shortest-path metric by DSD improves the performance of classical function prediction methods across the board.     

A mammalian spot test: induction of genetic alterations in pigment cells of mouse embryos with x-rays and chemical mutagens.

Embroys heterozygous for five recessive coat-color genes from the cross C 57 BL/6 J Han x T-stock were x-irradiated with 100/r o r treated in utero with 50 mg/3 kg methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), respectively. Controls consisted of irradiated embryos of C 57 BL x C 57 BL matings homozygous wild-type for the genes under study, and non-treated offspring of both types of mating. The colors of the spots were observed in the adult fur were either due to expression of the recessive coat genes or were white. I. Irradiated and mutagen-treated offspring of C 57 BL x T-stock matings had almost exclusively nonwhite spots, distributed randomly over the mouse surface. 2. Irraidated offspring of C 57 BL x C 57 BL matings had only white spots which were always midventral. 3. In non-treated offspring of both types of mating no spot could be observed. After correcting for white midventral spots observed in the one type of control, the frequency of expression of one or the other of the recessive color genes is calculated to be about 11% for embryos irradiated with 100r or 101/2 days postconception, about 1% for embryos irradiated with 100r at 9 days postconception, about &% for embryos treated with 50 mg MMS/kg at 101/2 days postconception, and about 8% for embryos treated with 50 mg EMS/2 days postconception. It is discussed that the white midventral spots are preferentially the result of pigment cell killing, while the nonwhite spots are preferentially the result of gene mutations or recombinational processes like mitotic crossing over and mitotic gene conversion. Of numerical and structural chromosome aberrations only those come into question which are able to pass the filter of several mitoses. Therefore, the test system described is supposted to cover not only heitable DNA-alterations, but the whole spectrum of them.     

Characterization of binding properties of the myelin-associated glycoprotein to extracellular matrix constituents.

The myelin-associated glycoprotein (MAG) can be obtained from adult mouse brain from detergent-lysates of a crude membrane fraction as a 96-100 kd form (detergent solubilized MAG), and from 100,000 g supernatants of homogenates as a 90-96 kd form (soluble MAG). The soluble form distributes into the Triton X-114-poor aqueous phase, while detergent-solubilized MAG predominantly enters the Triton X-114-rich phase. Both molecular forms bind to heparin in hypo- and isotonic buffers. Soluble MAG binds to several collagens (type G, I, II, III, IV, V, VI, IX) with a kd of 5.7 X 10(-8) M for collagen type IX and 2.0 X 10(-7) for collagen type IV. Binding of 125I-labeled MAG to collagen G can be completely inhibited by unlabeled MAG and collagen G, but not by heat-denatured collagen. MAG does not bind to itself, laminin, fibronectin, or the neural cell adhesion molecules L1 and N-CAM. Binding of MAG to collagen G is most effectively blocked by a high molecular weight dextran sulfate, heparan sulfate and heparin, with chondroitin sulfate and a low molecular weight dextran sulfate being less potent blockers. These findings are in agreement with previous observations on the localization of MAG in basal lamina and interstitial collagens of the sciatic nerve in situ.     

In focus: Film,focus groups and working children in Bangladesh

The use of visuals in anthropological research is an established though much debated practice, both as a research tool and as a means of reporting. Pile sorts, mapping, thematic drawing, photographs, visual scales and pictorial triad testing are all visual methods that have been used in participatory and conventional ethnographic research to encourage discussion among study participants and to clarify detail. Our experience in the use of visual tools in a study conducted in 1997–98, among former child garment workers in Bangladesh, reinforces the value of the use of visuals in research. A documentary film was used in focus groups with children, most aged 10–13. The results suggest that film is a powerfully evocative tool and, combined with focus groups, is an excellent qualitative research technique. The research experience in Bangladesh also suggests that children are able to participate meaningfully in the research not in spite of but because of the use of documentary film.     

Codon-usage variants in the polymorphic (GGN) n trinucleotide repeat of the human androgen receptor gene

The human androgen receptor gene (hAR) has a long, polymorphic trinucleotide (GGN; glycine) _n repeat in the 3′ portion of its first exon, with n = 10–31. Owing to technical difficulties that have precluded routine sequencing of this region, it is widely unknown that N represents T, G or C, and that the usual sense codon sequence of the GGN tract is (GGT)₃GGG(GGT)₂(GGC)_4–25. Furthermore, on 4 of 61 X chromosomes, we observed that the internal GGT sequence was present three or four times instead of twice. Strikingly, each of the three alleles with an internal (GGT)₃, and only these three, also had a (GGC)₂₀ repeat. The size or composition of a (GGN) _n repeat was not correlated with the length of the accompanying (CAG) _n CAA repeat in the 5′ portion of exon one. Hence, codon-usage variants of the GGN tract may be used to seek associations with particular diseases, as diagnostic aids in families with androgen insensitivity whose AR mutations have not yet been identified, or as internal controls for observations on intergenerational contractions or expansions of the (CAG) _n CAA tract in a given hAR allele. Received: 28 May 1997 / Accepted: 22 July 1997     

Serum levels of marine-derived n-3 fatty acids in Icelanders, Japanese, Koreans, and Americans--a descriptive epidemiologic study

In the 1990s Iceland and Japan were known as countries with high fish consumption whereas coronary heart disease (CHD) mortality in Iceland was high and that in Japan was low among developed countries. We described recent data fish consumption and CHD mortality from publicly available data. We also measured CHD risk factors and serum levels of marine-derived n-3 and other fatty acids from population-based samples of 1324 men in Iceland, Japan, South Korea, and the US. CHD mortality in men in Iceland was almost 3 times as high as that in Japan and South Korea. Generally, a profile of CHD risk factors in Icelanders compared to Japanese was more favorable. Serum marine-derived n-3 fatty acids in Iceland were significantly lower than in Japan and South Korea but significantly higher than in the US.     

Fold recognition and accurate sequence-structure alignment of sequences directing beta-sheet proteins

The ability to predict structure from sequence is particularly important for toxins, virulence factors, allergens, cytokines, and other proteins of public health importance. Many such functions are represented in the parallel beta-helix and beta-trefoil families. A method using pairwise beta-strand interaction probabilities coupled with evolutionary information represented by sequence profiles is developed to tackle these problems for the beta-helix and beta-trefoil folds. The algorithm BetaWrapPro employs a "wrapping" component that may capture folding processes with an initiation stage followed by processive interaction of the sequence with the already-formed motifs. BetaWrapPro outperforms all previous motif recognition programs for these folds, recognizing the beta-helix with 100% sensitivity and 99.7% specificity and the beta-trefoil with 100% sensitivity and 92.5% specificity, in crossvalidation on a database of all nonredundant known positive and negative examples of these fold classes in the PDB. It additionally aligns 88% of residues for the beta-helices and 86% for the beta-trefoils accurately (within four residues of the exact position) to the structural template, which is then used with the side-chain packing program SCWRL to produce 3D structure predictions. One striking result has been the prediction of an unexpected parallel beta-helix structure for a pollen allergen, and its recent confirmation through solution of its structure. A Web server running BetaWrapPro is available and outputs putative PDB-style coordinates for sequences predicted to form the target folds.