Quantifying the increase in average human heterozygosity due to urbanisation.

The human population is undergoing a major transition from a historical metapopulation structure of relatively isolated small communities to an outbred structure. This process is predicted to increase average individual genome-wide heterozygosity (h) and could have effects on health. We attempted to quantify this increase in mean h. We initially sampled 1001 examinees from a metapopulation of nine isolated villages on five Dalmatian islands (Croatia). Village populations had high levels of genetic differentiation, endogamy and consanguinity. We then selected 166 individuals with highly specific personal genetic histories to form six subsamples, which could be ranked a priori by their predicted level of outbreeding. The measure h was then estimated in the 166 examinees by genotyping 1184 STR/indel markers and using two different computation methods. Compared to the value of mean h in the least outbred sample, values of h in the remaining samples increased successively with predicted outbreeding by 0.023, 0.038, 0.058, 0.067 and 0.079 (P<0.0001), where these values are measured on the same scale as the inbreeding coefficient (but opposite sign). We have shown that urbanisation was associated with an average increase in h of up to 0.08-0.10 in this Croatian metapopulation, regardless of the method used. Similar levels of differentiation have been described in many populations. Therefore, changes in the level of heterozygosity across the genome of this magnitude may be common during isolate break-up in humans and could have significant health effects through the established genetic mechanism of hybrid vigour/heterosis.

Effects of isolation and inbreeding on human quantitative traits: an example of biochemical markers of hemostasis and inflammation.

Isolation is a known force in evolutionary biology and one of the main factors in speciation. One of the main consequences of severe isolation is reduced mate choice, which results in the occurrence of inbreeding as a result of isolation. We investigated the effects of individual genome-wide heterozygosity measured as the multilocus heterozygosity (MLH) on biochemical markers of hemostasis and inflammation in 1,041 individuals from the island of Vis, Croatia, where inbreeding is prevalent and a wide range of variation in the genome-wide heterozygosity is expected. Assessment of individual genome-wide heterozygosity was based on genome-wide scans using 800 microsatellite (STR) and 317,503 single nucleotide (SNP) polymorphic markers in each examinee. In addition, for each examinee we defined a personal genetic history (PGH) based on genealogical records. The association between PGH and MLH and fibrinogen, D-dimer (Dd), von Willebrand factor (vWF), tissue plasminogen activator (tPA), and C-reactive protein (CRP) was performed with a mixed model, controlling for possible confounding effects. PGH was a significant predictor only for tPA (P < 0.001), whereas neither of the two MLH measures exhibited significant association with any of the investigated traits. The effects of individual genome-wide heterozygosity are most likely expressed in highly polygenically determined traits or in traits that are mediated by rare and recessive genetic variants. Weak associations between PGH and MLH and markers of hemostasis and inflammation suggest that their genetic control may not be highly polygenic and that they could be promising targets for genetic association studies.

Effects of genome-wide heterozygosity on a range of biomedically relevant human quantitative traits.

The dramatic changes in human population structure over the last 200 years have resulted in significant levels of outbreeding, which, in turn, is predicted to lead to increased levels of individual genetic diversity (genome-wide heterozygosity, h). To investigate possible effects of these large demographic changes on global health, we studied the effect of h, measured as relative heterozygosity, h(R), on 15 disease-related traits in four groups of individuals with widely differing ancestral histories (ranging from outbred to inbred) from the Dalmatian islands in Croatia. Higher levels of h(R), estimated using 1184 STR/indel markers, were found in the outbred group (P < 0.0001) and were associated with lower blood pressure (BP) and total/LDL cholesterol (P = 0.01 and 0.01, respectively) after controlling for other factors, with BP showing a strong sex effect (males P > 0.5 and females P = 0.002). These findings, if replicated, suggest that h(R) be considered as a genetic risk factor in genetic epidemiological studies on common disease traits. They are consistent with the well-known effects of heterosis (hybrid vigour) described when outcrossing animals and plants. Outbreeding resulting from urbanization and migration from traditional population subgroups may be leading to increasing h(R) and may have beneficial effects on a range of traits associated with human health and disease. Other traits, such as age at menarche, IQ and lifespan, which have been changing during the decades of urbanization, may also have been influenced by demographic factors.

Strategy for mapping quantitative trait loci (QTL) by using human metapopulations.

AIM: To present a novel strategy for mapping quantitative trait loci (QTL), using human metapopulations. The strategy is based on the expectation that in geographic clusters of small and distinct human isolates, a combination of founder effect and genetic drift can dramatically increase population frequency of rare QTL variants with large effect. In such cases, the distribution of QT measurements in an (affected) isolate is expected to deviate from that observed in neighboring isolates. METHODS: We tested this hypothesis in 9 villages from a larger Croatian isolate resource, where 7 Mendelian disorders have been previously reported. The values of 10 physiological and biochemical QTs were measured in a random sample of 1001 individuals (100 inhabitants of each of 9 villages and 101 immigrant controls). RESULTS: Significant over- or under- representation of individuals from specific villages in extreme ends of standardized QT measurement distribution was found 10 times more frequently than expected by chance. The large majority of such clusters of individuals with extreme QT values (34/36, 94.4%) originated from the 6 villages with the most pronounced geographic isolation and endogamy. CONCLUSION: Early epidemiological assessment supports the feasibility of the proposed strategy. Clusters of individuals with extreme QT values responsible for over-representation of single villages can usually be linked to a larger pedigree and may be useful for further QTL mapping, using linkage analysis.

The power to detect disease associations with mitochondrial DNA haplogroups.

Genetic variation of mitochondrial DNA (mtDNA) has been linked to a number of multifactorial diseases, but there is currently no tool available to predict the optimal size for these investigations. We used a simulation-based (Monte Carlo) permutation test to generate power curves for European mtDNA haplogroup studies, to derive a universal equation to enable power calculations for prospective studies across the globe, and to show that very large cohorts are required to reliably detect an association with complex human diseases. In some populations, geographical variation in haplogroup frequencies will prevent the reliable detection of subtle haplogroup associations with uncommon disorders.

Increased level of linkage disequilibrium in rural compared with urban communities: a factor to consider in association-study design.

Few studies have investigated genetic differentiation within nonisolate European populations, despite the initiation of large national sample collections such as U.K. Biobank. Here, we used short tandem repeat markers to explore fine-scale genetic structure and to examine the extent of linkage disequilibrium (LD) within national subpopulations. We studied 955 unrelated individuals of local ancestry from nine Scottish rural regions and the urban center of Edinburgh, as well as 96 unrelated individuals from the general U.K. population. Despite little overall differentiation on the basis of allele frequencies, there were clear differences among subpopulations in the extent of pairwise LD, measured between a subset of X-linked markers, that reflected presumed differences in the depths of the underlying genealogies within these subpopulations. Therefore, there are strategic advantages in studying rural subpopulations, in terms of increased power and reduced cost, that are lost by sampling across regions or within urban populations. Similar rural-urban contrasts are likely to exist in many other populations with stable rural subpopulations, which could influence the design of genetic association studies and national biobank data collections.

Mapping quantitative trait loci using linkage disequilibrium: marker- versus trait-based methods.

Two approaches for mapping quantitative trait loci (QTL) using linkage disequilibrium at the population level were investigated. In the trait-based (TB) approach, the frequencies of marker alleles (or genotypes) are compared in individuals selected from the two tails of the trait distribution. The TB approach uses phenotypic information only in the selection step. In the marker-based (MB) approach, the quantitative trait values for the marker genotypes in the selected individuals are compared. The MB approach uses both the difference in marker allele (or genotype) frequencies and the phenotypic values of each marker genotype in the selected samples. We quantify the power of each approach and show that the power of the MB approach is greater than or equal to that of the TB approach. The advantage of the former is expected to increase with increasing number of traits phenotyped. Our accurate predictions obviate the need for elaborate simulation studies.

Transcriptome analysis of human autosomal trisomy.

We present transcriptome analyses of primary cultures of human fetal cells from pregnancies affected with trisomy 21 (t21) and trisomy 13 (t13). Pooled mRNA samples from t21 and t13 cases were used for comparative hybridizations to cDNA arrays with pooled mRNA from normal cells. When the array cDNAs were grouped by chromosomal location the relevant trisomic chromosome could be clearly identified as showing the most significant misregulation. The average level of transcription on the trisomic chromosome was increased only approximately 1.1-fold compared to normal cells on array analysis. Since the karyotype could be accurately predicted by the transcriptome this could provide a novel method of detecting aneusomy of unknown position. Subsequent analysis of individuals cases demonstrated that variation in transcriptional profiles between samples within each class made transcriptional karyotyping difficult without pooling or the use of arrays with a higher proportion of all human cDNAs. Interestingly, consistent differences in the relative expression levels between chromosomes were detected suggesting that genomic control mechanisms may act over larger distances than previously thought. Most (>95%) >+/-2 SD misregulated genes did not map to the trisomic chromosome and significant misregulation was more common in t13 than t21. These data support a model of a subtle primary upregulation of genes on the trisomic chromosome resulting in a secondary, generalized and more extreme transcriptional misregulation. It seems likely that the degree of this misregulation determines the severity of the phenotype in most aneuploidy.

Evidence for an age-related influence of microsatellite instability on colorectal cancer survival.

It is well established that microsatellite instability (MSI), the hallmark of defective DNA mismatch repair (MMR), is associated with prolonged survival in colorectal cancer compared with tumours that are microsatellite stable (MSS). MSI in sporadic colorectal tumours is primarily due to epigenetic silencing of MLH1. However, there are no prospective population-based studies of survival in patients with germline MMR gene mutations who develop cancer. Although MSI is almost universal in tumours from HNPCC family members, there is a potential confounding effect of ascertainment and other biases that could explain the apparent survival benefit in HNPCC families. Resolving whether germline MMR gene mutations impact on survival is important because it potentially undermines the rationale for surveillance of mutation carriers. Here, we report an investigation of the influence of MSI on survival in cohorts of cancer patients (aged < 30 years at diagnosis, n = 118; non-age-selected, n = 181) in the context of clinicopathologic variables. There was a substantial age-related influence of tumour MSI status on survival. In young patients with tumour MSI, 65% of patients with MSI tumours had germline MSH2 or MLH1 mutations. Clinicopathologic variables and tumour MSI of the cohort were studied with respect to survival and compared with control groups. Young patients had excess MSI tumours (p < 0.000001), mucinous tumours (p < 0.01), advanced disease (p approximately 0.001) and poorer 5-year survival compared with older cases. Cox proportional hazard analysis identified Dukes' stage, age at diagnosis and calendar year of treatment as independent predictors of survival. There was no detectable association between tumour MSI and survival in young patients, although we confirmed previous observations that MSI is associated with better prognosis in later onset cohorts. These findings underscore the rationale for surveillance and early identification of tumours in MMR gene carriers as well as refining understanding of the influence of MSI on cancer progression.