Epigenetics,heritability and longitudinal analysis

Background

Longitudinal data and repeated measurements in epigenome-wide association studies (EWAS) provide a rich resource for understanding epigenetics. We summarize 7 analytical approaches to the GAW20 data sets that addressed challenges and potential applications of phenotypic and epigenetic data. All contributions used the GAW20 real data set and employed either linear mixed effect (LME) models or marginal models through generalized estimating equations (GEE). These contributions were subdivided into 3 categories: (a) quality control (QC) methods for DNA methylation data; (b) heritability estimates pretreatment and posttreatment with fenofibrate; and (c) impact of drug response pretreatment and posttreatment with fenofibrate on DNA methylation and blood lipids.

Results

Two contributions addressed QC and identified large statistical differences with pretreatment and posttreatment DNA methylation, possibly a result of batch effects. Two contributions compared epigenome-wide heritability estimates pretreatment and posttreatment, with one employing a Bayesian LME and the other using a variance-component LME. Density curves comparing these studies indicated these heritability estimates were similar. Another contribution used a variance-component LME to depict the proportion of heritability resulting from a genetic and shared environment. By including environmental exposures as random effects, the authors found heritability estimates became more stable but not significantly different. Two contributions investigated treatment response. One estimated drug-associated methylation effects on triglyceride levels as the response, and identified 11 significant cytosine-phosphate-guanine (CpG) sites with or without adjusting for high-density lipoprotein. The second contribution performed weighted gene coexpression network analysis and identified 6 significant modules of at least 30 CpG sites, including 3 modules with topological differences pretreatment and posttreatment.

Conclusions

Four conclusions from this GAW20 working group are: (a) QC measures are an important consideration for EWAS studies that are investigating multiple time points or repeated measurements; (b) application of heritability estimates between time points for individual CpG sites is a useful QC measure for DNA methylation studies; (c) drug intervention demonstrated strong epigenome-wide DNA methylation patterns across the 2 time points; and (d) new statistical methods are required to account for the environmental contributions of DNA methylation across time. These contributions demonstrate numerous opportunities exist for the analysis of longitudinal data in future epigenetic studies.

     

Role of the kallikrein-kinin system in glandular secretion

Microsomes were prepared from liver, lung and kidney of rats and rabbits using a Ca⁺² aggregation method. Microsomal protein yield from the lung of both species was higher by this method of preparation as compared with ultracentrifuged samples. Specific activities of rat and rabbit pulmonary p-chloro-N-methylaniline (CMA) demethylase, biphenyl 4-hydroxylase and rat pulmonary TPNH cytochrome c reductase also were decreased. Specific activities of rabbit hepatic TPNH cytochrome c reductase, CMA N-demethylase, UDP-glucuronyltransferase and biphenyl hydroxylase were decreased by calcium aggregation Renal enzyme activities were unchanged by this method of preparation. These data indicate an apparent species and organ difference in microsomal enzyme response to calcium aggregation.     

The immunological similarity of rat glandular kallikreins.

The immunological properties of the submandibular kallikrein, urinary kallikrein, pancreatic kallikrein and pancreatic prekallikrein of the rat were studied by immunodiffusion, immunoelectrophoresis and radioimmunoassay. Although they behaved differently electrophoretically, all the antigens showed identical immunological behaviour. The implications of this are discussed.     

Rapid purification of a prekallikrein from rat pancreas

A prekallikrein from rat pancreas was purified 1500-fold with an overall yield of 20% using a rapid, simple procedure. DEAE-Sephadex A-50 chromatography permitted the separation of two prekallikrein components present in rat pancreatic homogenates; the major fraction was further purified by Sephadex G-100 gel filtration and immunoadsorption chromatography. The zymogen is a single-chain molecule with pI 4·35. Apparent M_r values of 38,000 and 37,000 were determined by sodium dodecyl sulfate-polyacrylamide gradient electrophoresis and gel filtration, respectively.     

Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro

By employing the principles of "activated swelling", monosized, superparamagnetic polymer particles have been prepared ranging in size from 1-100 microns. Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes. Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies. So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood. These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas. High speed and specificity are the most conspicuous features of immunomagnetic cell separation. These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens. Both higher sensitivity and specificity have been obtained. The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions. The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision. The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail. Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process. Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.     

Immunohistochemical localization of kallikrein in human pancreas and salivary glands

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

CA 125--epitopes and molecular size

CA 125 has two main immunogenic areas reactive with mouse and rat monoclonal antibodies. These areas are defined by the antibodies OC 125 and M11, respectively. Antibodies related to the main groups are named OC 125-like and M 11-like antibodies. The OC 125-like antibodies can be subgrouped into four sets, whereas the M 11-like antibodies segregate into many closely related binding specificities. One M 11-like antibody, ZR38, is not fully inhibited by any other M 11-like antibody and therefore represents a distinct subgroup. A single antibody, OV 197, is related to some OC 125-like antibodies, but not to OC 125. However, OC 125 enhances binding of OV 197 to its epitope. A new antibody, 7C 12, induces conformation changes, affecting binding of some M 11-like and some OC 125-like antibodies. CA 125 exists as very large molecules that can be partly disrupted by SDS and heat. The form found in serum might be a degradation product from a larger molecule found in the abdominal and other serous cavities.