Within individuals, many blood cell measurements remain stable longitudinally during periods of health. A balance between hemopoiesis, sequestration and apoptosis ensures that an individual's blood cell concentration is maintained within a narrow range of physiological values. In contrast, there is considerable variation in the number and size of blood cells between individuals. Whilst molecular techniques have identified a myriad of genes involved in hemopoiesis and blood cell apoptosis- particularly in pathological and inflammatory conditions- they have provided little information as to the genetic causes of variation between healthy individuals. A knowledge of these genes would be important not only theoretically, but may also identify novel therapeutic targets in pathological states characterized by abnormal blood cell counts.
The aim of this thesis was to investigate the genetic causes of variation in blood cell
concentrations. 332 monozygotic and 487 dizygotic twin pairs were measured longitudinally at twelve, fourteen and sixteen years of age on a variety of blood cell variables including: hemoglobin, red blood cell count, hematocrit, mean corpuscular volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, total white cell count, level of neutrophils, monocytes, eosinophils, total lymphocytes, CD3+ lymphocytes, CD4+ lymphocytes, CD8+ lymphocytes, CD 19+ lymphocytes, Natural Killer cells and CD4+/CD8+ ratio. Structural equation models were fitted to the data in order to estimate the relative importance of genetic and environmental factors, and also to investigate more complicated questions such as whether there were sex differences in the magnitude of genetic and environmental effects, whether covariation between blood cell measures was due to the same or different genes, and whether the same or different genes affected the phenotypes over time. The twins, their
parents, and siblings were typed for 757 highly polymorphic microsatellite markers spaced across the genome at an average intermarker distance 4.6 cM. Genome-wide linkage analyses were performed in order to identity chromosomal location of quantitative trait loci (QTLs) influencing the measures. Finally, the power of a multivariate variance components test of linkage was explored analytically and via simulation, before being applied to regions of suggestive linkage in an attempt to increase the power to detect these loci.
Genetic factors contributed strongly to most blood cell variables accounting for between 38 and 95 percent of the phenotypic variance in these measures. In contrast, the impact from common environmental factors tended to be small or not significant, save in the case of mean cell hemoglobin concentration which demonstrated little evidence of genetic influence. Several variables displayed sex differences in the magnitude of genetic and
environmental effects, most notably neutrophil and eosinophil count, although there was no evidence that different genes affected the traits in males and females.
Structural equation modelling indicated the existence of a pleiofropic genetic factor affecting the red cell indices (i.e. hemoglobin, red cell count, mean corpuscular volume), a genetic factor affecting neutrophil and monocyte counts, and a genetic factor influencing the lymphocyte subsets. The analyses also revealed genetic factors which were specific to each trait. Longitudinal analyses suggested that the majority of genetic variance was transmitted from age twelve to ages fourteen and sixteen. Most variables also demonstrated significant genetic innovations at ages fourteen and sixteen, suggesting that sex hormones secreted during puberty induced the expression of "new" genes which affected these variables. Bivariate longitudinal analyses indicated that the same simplex process
underlay much of the genetic covariation between the red cell indices, between neutrophil and monocyte count, and between the CD4+ and CD8+ T lymphocytes.
Genome wide linkage analyses revealed strong evidence for linkage in the 2q33 region for eosinophil count and the 9p24.1 region for total white cell count. Candidate genes in the 2q33 region included CD28 and CTLA-4 (involved in T-cell signalling) and Caspase-8 (involved in cell apoptosis). Several other regions of "suggestive" linkage were also identified, most notably 11pl15.1 - 11p14 for CD4- CD8 ratio, and 19q13.13 - 19q13.31 for platelet count.
The power of multivariate variance components linkage analysis depended upon the size and source of the residual correlation between phenotypes. In particular, the power to detect a pleiotropic QTL was greatest when the QTL produced covariation in the direction opposite to the unique environment. Applying the multivariate
procedure to the longitudinal data from platelet count and CD4/CD8 ratio increased the evidence for linkage in the suggestive regions.