Genetic determinants of hemostasis phenotypes in Spanish families.

BACKGROUND: Recent studies have described genetic mutations that affect the risk of thrombosis as a result of abnormal levels of such hemostatic parameters as protein C, protein S, and the activated protein C resistance ratio. Although these mutations suggest that genes play a part in determining variability in some hemostasis-related phenotypes, the relative importance of genetic influences on these traits has not been evaluated. METHODS AND RESULTS: The relative contributions of genetic and environmental influences to a panel of hemostasis-related phenotypes were assessed in a sample of 397 individuals in 21 extended pedigrees. The effects of measured covariates (sex, age, smoking, and exogenous sex hormones), genes, and environmental variables shared by members of a household were quantified for 27 hemostasis-related measures. All of these phenotypes showed significant genetic contributions, with the majority of heritabilities ranging between 22% and 55% of the residual phenotypic variance after correction for covariate effects. Activated protein C resistance ratio, activated partial thromboplastin time, and Factor XII showed the strongest heritabilities, with 71.3%, 83.0%, and 67.3%, respectively, of the residual phenotypic variation attributable to genetic effects. CONCLUSIONS: These results clearly demonstrate the importance of genetic factors in determining variation in hemostasis-related phenotypes that are components of the coagulation and fibrinolysis pathways and that have been implicated in risk for thrombosis. The presence of such strong genetic effects suggests that it will be possible to localize previously unknown genes that influence quantitative variation in these hemostasis-related phenotypes that may contribute to risk for thrombosis.

Comparison of biochemical polymorphisms and short tandem repeat (STR) DNA markers for paternity testing in rhesus monkeys (Macaca mulatta).

Genetic markers are indispensable for molecular and statistical genetic research involving nonhuman primates. Genetic markers must be used to ascertain parentage and to confirm the accuracy of pedigrees based solely on housing or demographic records; otherwise, the results of pedigree, linkage, or quantitative genetic analyses may be unreliable. Until recently, most genetic markers used in nonhuman primates were plasma proteins or isozyme polymorphisms, which were required in large numbers, because levels of genetic variation revealed by these markers were rather low. We compared the newer, PCR-amplified short tandem repeat markers (STRs) with a panel of classical biochemical polymorphic markers, for paternity determination among captive-bred rhesus monkeys. The STR markers exhibited an average genetic diversity of 64% and an expected paternity exclusion probability of 0.443. Both of these were greater than the average 54.5% genetic diversity and 0.298 exclusion probability exhibited by the biochemical markers. The STRs were much more efficient than the biochemical markers for parentage determination, since they required only half the amount of genetic typing data to resolve an average paternity case. Thus, the results of applying these two classes of genetic markers in paternity tests were somewhat different than expected on the basis of theoretical exclusion probabilities. These differences were probably due to inbreeding and other genetic differences among breeding colonies. Because they are more informative and provide rapid and efficient genetic data, STRs are now the method of choice for parentage determination and pedigree corroboration among nonhuman primates.

Absence of a significant mixed lymphocyte reaction in a marsupial (Monodelphis domestica).

Previously we reported preliminary results suggesting that the marsupial Monodelphis domestica fails to exhibit a mixed lymphocyte reaction with allogeneic lymphocytes. To test whether this observation is simply a matter of a response too weak to detect, but capable of being augmented by immunization, we performed mixed lymphocyte culture tests on 23 of these animals that had been immunized with lymphocytes. Despite the fact that all recipients were sensitized to the lymphocytes of the donors, none of the animals had a substantial mixed lymphocyte response. Significant stimulation was noted with the mitogen concanavalin A; thus, the T cells were immunologically competent. It seems likely that the failure of this species to exhibit a significant mixed lymphocyte response is due to T cells whose ontogeny differs from that of the T cells of eutherian mammals.

Fate of allogeneic skin transplantations in a marsupial (Monodelphis domestica).

Previously we reported that the South American gray short-tailed opossum, Monodelphis domestica, had an MHC class-I locus similar to that of eutherian species. In addition to the detection of lymphocyte antigens by cytotoxic antisera, we concluded that this marsupial rejected allogeneic skin grafts, as would be expected of animals with MHC class-I polymorphism. However, this conclusion was based on a limited number of skin transplants that were assayed for only a short period. Here we report the results of 22 reciprocal skin grafts made between individuals of known genetic relationships. On the basis of gross inspection of the grafts and histologic examination, we found that the average time of the onset of graft rejection was about 19 days and that the average time for complete graft rejection was about 31 days. In general, it took longer for the onset of graft rejection among pairs of genetically related animals than among less related animals. These results indicate unequivocally that this marsupial species has a high degree of class-I polymorphism and rejects allogeneic skin transplants in a manner similar to but more slowly than eutherian mammals.

Maintenance of genetic variability in a specific pathogen-free breeding colony.

We used 18 genetic loci including blood groups, isozymes, and a serum protein to evaluate our efforts to preserve genetic variability in a specific pathogen-free (SPF) colony of rhesus monkeys. We compared genetic variability in the SPF population to the virally contaminated, non-SPF population from which it was derived. There was no change in the average gene diversity between the SPF and non-SPF populations. However, gene diversity at blood group Q locus increased significantly in the SPF population, while blood group M locus showed an insignificant trend toward decreased gene diversity. Allele frequencies changed significantly at blood group Q locus, although no alleles were lost from the population. We hypothesized that this change was due to extensive overreproduction by a small number of founder males that possessed the initially rare allele, Q1. There was no evidence that this change was associated with genes involved in viral infection.

Effect of radiation and age on immunoglobulin levels in rhesus monkeys (Macaca mulatta).

We report the results of a study on the immunoglobulin levels of rhesus monkeys (Macaca mulatta) in a colony consisting of the survivors of monkeys that received a single whole-body exposure to protons, electrons or X rays between 1964 and 1969. This colony has been maintained to assess the long-term effects of ionizing radiation on astronauts and high-flying pilots. Of the original 358 monkeys that were retained for lifetime studies, 129 (97 irradiated and 32 controls) were available for our study. We found no significant difference between the irradiated and control monkeys in mean levels of IgA, IgG and IgM, irrespective of the radiation treatment. The availability of these aged monkeys provided a unique opportunity to compare their immunoglobulin levels to those of other monkeys of various ages, and thus assess the effect of age on immunoglobulin levels. We found that only the IgA levels increase with age.

A simple method for genetic typing of transferrins in nonhuman primates.

The serum protein transferrin (Tf) is a valuable marker for genetic studies of primates, because it is usually polymorphic, exhibiting as many as 13 allelic forms with high heterozygosity. The standard procedure to detect the different phenotypes requires vertical electrophoresis on polyacrylamide gels for 18 h at 4 degrees C. We have simplified the procedure by using the automated programmable PhastSystem for electrophoresis, which uses precast miniature gels and takes less than 2 h. Also, we have developed a pool of sera that displays the 6 most common alleles in rhesus monkeys. This pool allows for a more accurate assignment of phenotypes. Using this simplified procedure, we have typed over 3000 monkeys of various species and have confirmed the reliability of this technique for parentage determination.

The effect of age on the level of human ABO blood group antibodies.

It is generally assumed that cellular and humoral immunity decline in aging humans. Although there have been reports that the naturally-occurring ABO antibodies also decline with age, some of the data are incomplete and others contradictory. Our study involved only healthy women of various ages and included assays of anti-A and anti-B titers in A, B, and O subjects. Statistical analyses of the data showed that while the antibody titers decreased with age, the amount of change was very small relative to the amount of variability among individuals and the variability attributable to blood type and season of the year. Comparisons with a previous study (1) from our laboratory showed that the changes due to aging were also small relative to the variability in titer scores within an individual over the course of a year. Based on this study, we conclude that the decrease in antibody titers with age is of doubtful clinical significance.

Blood groups in a marsupial (Monodelphis domestica).

We produced alloimmune sera against erythrocytes of the marsupial, Monodelphis domestica. By absorption fractionation, six operationally monospecific blood typing reagents (hemolysins) were prepared. Using segregation analyses, we found that each reagent detects a blood group antigen controlled by an autosomal dominant gene. With the description of these genetic markers, the utility of M. domestica as an animal model is enhanced.

Linkage between two blood-group markers in rhesus monkeys (Macaca mulatta).

Using marker data on an extensive pedigreed population from the Wisconsin Regional Primate Research Center, we report the first genetic linkage in rhesus monkeys (Macaca mulatta). Five blood-group markers (G, I, J, K, and Q) and one serum protein marker (transferrin) were analyzed. Linkage analyses indicate the existence of loose linkage between G and Q (maximum lod score, Zmax = 4.84 at theta m = 0.314 +/- 0.38, theta f = 0.266 +/- 0.054). The genetic linkage described here constitutes the initial step toward the description of a genetic map in this species.

Effects of chronic blood loss in a marsupial (Monodelphis domestica).

Monodelphis domestica, the gray short-tailed opossum, is used increasingly as an animal model in studies that require repeated blood sampling. Consequently, it is important to establish safe bleeding regimens. We investigated the effects of repeated blood loss on various hematologic values and on different organs in this species. Approximately 2 ml of blood were taken weekly from 20 animals for 13 weeks. The animals were then necropsied; members of an age- and sex-matched control group were bled (2 ml) once and necropsied immediately to obtain baseline data. Ultimately, each animal in the experimental group lost approximately three times its total blood volume. After the first bleeding in the chronically bled group, the red blood cell counts, hemoglobin, and hematocrit values decreased significantly but remained constant thereafter. In another experimental group bled only once, the hematologic values rose to higher than baseline levels after a rest of 2 weeks. Thereafter the values slowly returned to baseline levels. A notable increase in Howell-Jolly bodies occurred in the chronically bled group. Histologically, there was marked erythroid hyperplasia in the bone marrow and extramedullary hematopoiesis in the spleen, but none in the liver. Because there were no obvious detrimental physiologic effects, we conclude that M. domestica is markedly tolerant of chronic blood loss.

Genetic structure of three populations of rhesus macaques (Macaca mulatta): Implications for genetic management.

One of the prime concerns at zoos and at primate breeding facilities is to maintain genetic variability. This can be accomplished by avoiding inbreeding. It is relatively easy to assess genetic variability and the level of inbreeding by using pedigree information and genetic markers. In this study we used genetic markers controlled by 6 independent polymorphic loci (GPI, PGD, CA2, MPI, DIA1, Tf) to ascertain genetic variation in two captive and one wild population of rhesus monkeys. Two other loci ADA and NP were also examined and found to be monomorphic in the three populations. F-statistics and contingency chi-square analyses indicated that there was significant genetic differentiation among the populations. We also found that the mean heterozygosities were very similar in the three populations, in spite of the diverse breeding strategies. These data are important because rhesus monkeys are frequently used for biomedical research; and the genetic markers provide useful information for genetic management of captive colonies of nonhuman primates. © 1992 Wiley-Liss, Inc.

Ontogeny of the thymus gland of a marsupial (Monodelphis domestica).

The gray short-tailed opossum (Monodelphis domestica) has recently been established as a laboratory animal; consequently, its utility as an animal model is enhanced as more of its basic biologic characteristics become defined. To this end, we studied the ontogeny of the thymus gland in M. domestica ranging in age from the day of birth to over three years. In contrast to most Australian marsupials, M. domestica possesses only a thoracic thymus. The thymus is large in neonates and remains large until young adulthood (six months of age). It has a well-defined cortex, medulla and Hassall's corpuscles. At about six months of age, the thymuses of most animals begin to atrophy, the cortex and medulla being replaced by fat. By 28 months of age, about 50% of animals have thymuses that are completely atrophied. Thus, the ontogeny of the thymus of M. domestica appears to be similar to that of eutherian mammals.

Cellular immune response of a marsupial, Monodelphis domestica.

Marsupials are interesting subjects for studies of comparative and developmental immunology because they separated from eutherian mammals over 100 million years ago and because the newborns are still in a fetal state. We studied cellular immunity in a fully pedigreed colony of the marsupial, M. domestica (commonly called the gray short-tailed opossum). Peripheral blood lymphocytes were separated on nylon wool columns into adherent cells bearing surface immunoglobulin (B cells) and nonadherent cells (T cells) recovered in the ratio of 1:3. Peripheral blood lymphocytes responded by proliferation to Con A and other mitogens. Nonadherent cells were responsive to Con A, but adherent cells were not. Peripheral blood lymphocytes were stimulated weakly or not at all by allogeneic or xenogeneic (mouse) cells in mixed lymphocyte culture. Despite the weak MLC response, which was not due to genetic homogeneity, allogeneic and xenogeneic tail skin grafts were rejected promptly. These data suggest that the cellular immune response of M. domestica is similar to that of eutherian mammals with the notable exception of weak MLC responses.

Changes in American A.I.D. practice during the past decade.

We conducted a survey of donor artificial insemination (AID) practices within the United States. The survey document was sent to 360 practitioners listed by national infertility organizations as performing AID. In addition, we queried 100 practices that had responded to a similar survey in 1978. AID was actually performed during the survey year by 167 of the 282 respondents. Relatively few practices (23%) inseminated more than 10 patients per month. Donor payments increased from a mean of $25 to $40 over that period. A significant increase in the use of frozen semen was found. A majority of practices (52%) used a combination of basal body temperature and urinary LH to time inseminations. The fraction of practitioners who inseminated unmarried women increased substantially since the previous survey. The maximum number of pregnancies resulting from a single donor has not changed over the decade. However, on the average, a single donor is fathering fewer children. The percentage (39%) of practices that inseminate women because of genetic disease in the husband's family has remained about the same. Records of donors and AID children were maintained by 40% of the respondents, representing no change from the previous survey. Our data suggest that as many as 23,400 infants may have resulted from AID conceptions during the survey year. Further changes in the practice of AID can be expected as a result of the 1988 federal recommendations that all donor inseminations be undertaken with cryopreserved, quarantined semen.

Immune response of a marsupial (Monodelphis domestica) to sheep red blood cells.

A study of the humoral immune response of a marsupial, Monodelphis domestica, was performed using sheep red blood cells as the immunogen and a hemolytic titration as the assay. The primary response to a single intramuscular injection was similar to that observed in other mammalian species, except that the titers remained high for as long as 37 weeks after the initial injection. The secondary response was weaker than the primary response and diminished as early as 15 weeks after the initial injection (and only 11 weeks after the single booster injection). The hyperimmune response was similar to the secondary response, except that the titers remained consistent for up to 10 weeks and then fell to very low levels at 35 weeks. The data support the view that the primary humoral response of this marsupial is similar to that of eutherian mammals. However, the secondary and hyperimmune responses appear to be very weak, if present at all.