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 common feature of Mendelian diseases is that their characteristic phenotype is caused by a rare mutation in a single gene in the genome. Therefore, the segregation of affected individuals in families follows simple Mendelian predictions (1). The catalogue of known Mendelian diseases is regularly published, with some 8000 diseases or syndromes listed and new ones continually added to this number (2). The last decade saw great successes in identifying genetic variants underlying several thousands of these diseases (3-5). This success was facilitated by the fact that causal genetic mutation is both necessary and sufficient for the development of the disease, which is the key property of Mendelian diseases. This ensures good correlation between disease phenotypes and underlying genotypes (high “penetrance” and “detectance,” ie, the probabilities of observing the disease phenotype given the disease genotype, and vice versa), which is an important requirement for the success of gene mapping using pedigree-based approach (6,7).Most Mendelian diseases usually present at an early age and with a number of clinically apparent phenotypic changes. Such a spectrum of phenotypes, initially described as a distinct clinical syndrome, reflects the multiple roles the affected gene products have in human development and metabolism. As the human genome harbors some 25 000 predicted genes and an unknown number of conserved functional elements and regulatory regions, perhaps many more than 8000 Mendelian diseases should be expected. Many genes, however, may interact with each other within common biochemical pathways, thus limiting the number of possible phenotypic outcomes of their mutations. However, the diagnosis of Mendelian diseases is typically based on noticing visually apparent disease phenotypes.These phenotypes all have in common that they represent measurable human biological quantitative traits. Some of them (eg, blood pressure, body mass index, cholesterol levels, and blood glucose) have recently been identified by the World Health Organization as the main contributors to disease burden in developed countries (8). An understanding of their genetic regulation is therefore of great current interest (9,10). The genes underlying human quantitative traits (QT) may actually be easier to detect than those predisposing common complex diseases, as quantitative traits represent just a fraction of the many recognized risk factors underlying common complex diseases of late onset (11). In this paper, we present and test a novel strategy for finding very rare genetic variants with large effect on QTs in human populations, ie, genes underlying "invisible Mendelian diseases." The proposed approach relies on specific population genetic properties of geographically clustered and isolated human populations, often referred to as metapopulations, which allow for increased frequency of large effect genetic variants underlying quantitative trait distributions that would have extremely small frequencies in large outbred populations.Croatia has 15 Adriatic Sea islands with a population greater than 1000. The villages on the islands have unique population histories and have preserved their isolation from other villages and outside world through many centuries. The history, demography, and genetic structure of these villages have been investigated for more than 50 years. The research, mainly carried out by the Institute for Anthropological Research in Zagreb, Croatia, resulted in over 100 publications in international journals (12-14). On some of the islands, monogenic (Mendelian) diseases and rare genetic variants were found in unexpectedly high frequencies (15-27).

Table 1

Overview of the evidence of extremely rare mutations present in unusually high frequencies in specific Croatian island isolates

Association among education level, occupation status, and consanguinity in Tunisia and Croatia

Aim

To investigate the association between education level, occupation status (a proxy for socio-economic status), and consanguinity in 2 large data sets from Tunisia and Croatia countries with different attitudes toward consanguinity.

Methods

The sample of 1016 students, attending 5 university institutions in Monastir, Tunisia, were interviewed about the educational level and occupation status of their parents and the degree of parental relatedness. In Croatia, a sample of 1001 examinees from 9 isolated island populations was interviewed about their own educational level, occupation status, and consanguinity.

Results

Prevalence of consanguinity (offspring of second cousins or closer) among 1016 Tunisian students was 20.1%, and 9.3% among 1001 Croatian isolates. In Tunisia, the association between consanguinity and both parental degree of education and parental occupation status was highly significant in women (P<0.001), but not significant in men. In Croatia, no statistically significant associations were noted, although there was a consistent trend of increased prevalence of consanguinity with lower education level or occupation status in both genders, but more pronounced in women.

Conclusion

Association between education level, socio-economic status, and consanguinity needs to be taken into account in inbreeding studies in human populations. The relationship may be specific for each studied population and highly dependent on the cultural context. It is generally more pronounced among women in most settings.Consanguineous marriages are unions between two persons who share at least one recent common ancestor (1). In clinical genetics, a consanguineous marriage is commonly defined as union between subjects related as second cousin or closer, equivalent to an inbreeding coefficient in their progeny of F≥0.0156 (2). This kind of union was known to increase the risk of homozygous recurrence of deleterious recessive genes (3,4), and this could explain the increase of polygenic or multifactorially determined diseases in populations with high prevalence of consanguinity (1,5,6). A number of studies reported that offsprings of consanguineous parents had higher rates of neonatal, post-neonatal, child, and infant mortality than those of non-consanguineous parents (7-11).It is widely perceived that consanguinity is more prevalent among the underprivileged in the society (12-14). However, it is possible that factors that are not genetically determined, such as education level and socio-economic status of the subjects, have a confounding effect in the studies on consanguinity. To explore this, we investigated the association between education level, occupation status (a proxy for socio-economic status), and consanguinity in 2 large data sets available from Tunisia and Croatia. The analysis in these two countries with different attitudes toward consanguinity and causes and prevalence of inbreeding could reveal whether the presumed associations between education, occupation, and inbreeding could be generalized, or whether they are more complex and context-specific.     

Directing Diarrhoeal Disease Research towards Disease-burden Reduction

Despite gains in controlling mortality relating to diarrhoeal disease, the burden of disease remains unacceptably high. To refocus health research to target disease-burden reduction as the goal of research in child health, the Child Health and Nutrition Research Initiative developed a systematic strategy to rank health research options. This priority-setting exercise included listing of 46 competitive research options in diarrhoeal disease and their critical and quantitative appraisal by 10 experts based on five criteria for research that reflect the ability of the research to be translated into interventions and achieved disease-burden reduction. These criteria included the answerability of the research questions; the efficacy and effectiveness of the intervention resulting from the research; the maximal potential for disease-burden reduction of the interventions derived from the research; the affordability, deliverability, and sustainability of the intervention supported by the research; and the overall effect of the research-derived intervention on equity. Experts scored each research option independently to delineate the best investments for diarrhoeal disease control in the developing world to reduce the burden of disease by 2015. Priority scores obtained for health policy and systems research obtained eight of the top 10 rankings in overall scores, indicating that current investments in health research are significantly different from those estimated to be the most effective in reducing the global burden of diarrhoeal disease by 2015.Key words: Child heath, Diarrhoeal diseases, Mortality, Priority setting, Medical research     

Determining patient concerns before joint arthroplasty

Despite the prevalence of studies relating to hip and knee arthroplasties, no Canadian studies exist in the literature regarding patients' perceptions and concerns prior to undergoing these procedures. A 32-question, 5-point Likert scale questionnaire was developed and administered to a Canadian cohort. One hundred fifty-six knee and 85 hip arthroplasty patients returned the survey. Results consistently showed complications arising from surgery, ways to reduce pain after surgery, and ability to walk properly again as the 3 top concerns for patients whether stratified by age, sex, residential status or joint replacement type. Other results discussed include resources used by patients to become better informed about joint arthroplasty, differences in Western Ontario and McMaster Universities Index of Osteoarthritis scores between groups, and significant differences in patient concerns as determined by ridit analyses.     

Computer-assisted localization of osteoid osteoma: an initial experience

This article evaluates our initial experience with computer-assisted localization of osteoid osteoma. Nine patients with osteoid osteoma underwent minimally invasive computer-assisted surgery. Patients were followed prospectively for symptomatic relief and complications for an average of 31 months. Successful localization of osteoid osteoma occurred in 7 of 9 patients. Mean operative time was 88 minutes, and mean time to discharge was 1 day (range: same day to 2 days). No fractures, infections, or neurovascular complications occurred. Minimally invasive computer-assisted surgical excision of osteoid osteoma is a safe and feasible option for the surgical localization of osteoid osteoma. It is especially attractive for lesions located in poorly accessible anatomic sites.     

Genetic sub-structuring of Croatian island populations in the Southeastern European context: a meta-analysis

AimTo use the method of meta-analysis to assess the influence of island population isolation on the sub-structuring of the Croatian population, as well as the influence of regional population groups on the sub-structuring of the Southeastern European population with regard to basic population genetic statistical parameters calculated by using STR locus analysis.MethodsBio-statistical analyses were performed for 2877 unrelated participants of both sexes from Southeastern Europe. Nine autosomal STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S82) were analyzed by using standard F-statistics and population structure analysis (Structure software).ResultsGenetic differentiation of Croatian subpopulations assessed with the F_ST method was higher at the level of the Croatian population (0.005) than at the level of Southeastern Europe (0.002). The island of Vis showed the most pronounced separation in the Croatian population, and Albanians from Kosovo in the population of Southeast Europe, followed by Croatia, Bosnia and Herzegovina, and Hungary.ConclusionThe higher structure of Croatian subpopulations in relation to Southeastern Europe suggest a certain degree of genetic isolation, most likely due to the influence of endogamy within rural island populations.

The island populations of the eastern Adriatic have been the subject of multidisciplinary anthropological research for almost 50 years, starting with the pioneering work of Rudan et al in 1972 (1). A number of specific features of these rural populations has been revealed, which make them exceptional models for studying ethno-cultural, historical, migratory, and demographic characteristics of this region. More specifically, evolutionary forces (bottleneck effect and genetic drift) increase genome homogeneity within the genetic structure of such island isolates by eliminating certain genetic traits in favor of others and increasing the likelihood of finding low-impact alleles (2,3). The reduced genetic and environmental diversity makes genetically isolated populations suitable for the study of different complex and rare Mendelian hereditary diseases, since the combined action of genetic drift, inbreeding, and founder effect increases the prevalence of such diseases when compared with the general population.Southeastern Europe was one of Europe’s glacial refugia during the ice age, and the origin of postglacial resettlement of Europe in the Paleolithic and Neolithic. Due to this specific role and its position at the crossroads of migrations to and from Europe, this area was extensively investigated in the field of population genetics (4-7). Different genetic markers have been used to investigate the genetic landscape of Europe and determine the patterns of population sub-structuring at the regional and continental level (8). As a part of the comprehensive anthropological research on the population structure of Croatian island isolates, microsatellite DNA from different subpopulations has been previously analyzed to determine genetic diversity, population structure, and the degree of isolation of island populations (9). Similar studies were also conducted on a representative sample of the general Croatian population and other isolated populations from Southeastern Europe (10-12).This study represents a continuation of previous anthropogenetic research (6,13-16). We used statistical and analytical methods of meta-analysis to synthesize data from previously conducted, mutually independent studies of island and continental populations of Croatia and Southeastern Europe (Figure 1) based on analyses of autosomal STR markers, and data analyzed in this study for the first time.Open in a separate windowFigure 1Division of the sample into two hierarchical groups (left) and geographical position of the investigated Croatian subpopulations (right). NDI – North Dalmatian islands.The aim of this study was to determine the genetic characteristics of populations from Southeastern Europe, with special reference to Croatian island populations, and to investigate the effect of specific intrapopulation genetic structure on interpopulation relationships. Namely, a specific aim was to investigate the influence of island population isolation on the sub-structuring of the Croatian population, and the influence of regional population groups on the sub-structuring of Southeastern Europe with regard to basic population genetic statistical parameters calculated by using STR locus analysis.     

Influence of genetic substructuring of statistical forensic parameters on genetic short tandem repeat markers in the populations of Southeastern Europe

AimTo investigate the influence of specific intrapopulation genetic structures on interpopulation relationships. Special focus was the influence of island population isolation on the substructuring of the Croatian population, and the influence of regional population groups on the substructuring of Southeast European populations.MethodsAutosomal short tandem repeat (STR) loci were analyzed by using four forensic parameters: matching probability (PM), power of discrimination (PD), power of exclusion (PE), and polymorphic information content (PIC) on a sample of 2877 unrelated participants of both sexes. A sample set comprising 590 participants was analyzed for the first time, and 2287 participants were included from previous studies. The analysis was performed with PowerStats v. 1.2.ResultsThe analysis of forensic parameters for all nine loci in the Croatian subpopulations showed the largest deviations in the populations of the islands of Korčula and Hvar. The smallest deviations were found in the mainland population. As for Southeast European populations, the largest deviations were found in the population of North Macedonia, followed by Romania, Albanians from Kosovo, and Montenegro, while the smallest deviations were found in the population of Hungary.ConclusionThe comparison of forensic parameters between different subpopulations of Croatia and Southeast Europe indicates that the isolation of individual Croatian subpopulations and rare alleles in their gene pool affect the values of forensic parameters. Specific features of (sub)populations should be taken into account for appropriate sampling of the total population when creating a DNA database of STR markers.

Microsatellite markers are used in forensic research due to their high power of discrimination (PD) (usually >0.9, with observed heterozygosity >70%), position at separate chromosome locations (to avoid closely related loci), consistency and reproducibility of results after multiplication with other markers, and rare occurrences of stutter products (1). The use of short tandem repeat (STR) markers in paternity testing and forensic genetic analysis increases the PE from an initial 30% to 40% for blood typing, 80% for tissue typing, HLA analysis, 90% for combinations of HLA analysis with serological tests, and finally 99.99% for RFLP analysis to a minimum of 99.999% (2-4). In forensic research, the advantage of microsatellite markers is the possibility of simultaneous analysis of multiple loci in multiplex STR systems, which allows a high degree of individualization in identifying traces. In forensic applications where DNA is usually degraded, microsatellite markers of 100 to 400 bp are better markers than minisatellites (VNTRs) of 400 to 1000 bp (4).Microsatellite DNA differs for each person, but two people may have the same allelic variants at one or more STR loci. However, the probability of finding allelic variants in two individuals at, for example, 15 STR loci, analyzed using the PowerPlex 16 kit for the white population is 1.83 × 10¹⁷ (5). The average mutation rate for loci is variable, but its values are below 0.1%. Based on previous forensic research, the loci with the lowest mutation rate are CSF1PO, TH01, TPOX, D5S818, and D8S1179, while D21S11, FGA, D7S820, D16S539, and D18S51 have a significantly higher mutation rate (2,6-8).Specific population DNA databases serve to determine the genetic diversity of populations and to facilitate statistical calculations in forensic genetics. They are not based on individual DNA profiles, but the profiles represented in a particular population are used to determine allelic frequencies for further statistical calculations, ie, analysis of forensic identification parameters based on the assessment of PM, PD, PE, degree of loci heterozygosity (H), and likelihood ratio (LR). In order to form the most representative database of a certain population, it is important to investigate the genetic diversity of the population, its features, size, isolation, and the degree of genetic differentiation in relation to neighboring and distant populations (4).Therefore, the aim of this study was to investigate the existence of substructured subpopulations in Croatia, and the impact of their specific intrapopulation genetic structure on interpopulation relationships, and how these relationships affect the basic forensic statistical parameters of genetic STR markers.     

Two-dimensional gel electrophoresis of calf aqueous humor, serum, and filter-bound proteins

Recent studies have demonstrated that bovine and primate aqueous humor (AH) obstruct flow when perfused through artificial membranes with pore sizes similar to those found in the aqueous outflow pathway. Proteinaceous AH components were implicated in this phenomenon, which is not observed with serum diluted to comparable protein concentrations. In this study, we used two-dimensional gel electrophoresis to characterize the protein composition of calf AH and to identify those proteins binding to the filters and presumably causing this obstruction. Comparison of AH and serum under denaturing conditions showed quantitative and qualitative differences in their protein content. Among the most important: AH was seen to possess two protein subunit trains (approximately 28 kD and approximately 48 kD) not found in serum and two trains (approximately 28 kD and approximately 80 kD) with additionally charged components not found in serum. Serum, on the other hand, possesses one train (approximately 80-90 kD) not found in AH as well as a slightly greater relative amount of high-molecular weight protein subunits. The finding that hydrophobic filters retain more protein components than do hydrophilic filters suggests that the type and amount of protein adhering to them is determined largely by hydrophobic interactions. Whether such interactions occur in the outflow system, and if so, how they may relate to aqueous drainage remains to be determined.     

滇西地区农村老年人跟骨骨密度与ADL的关联研究

目的 分析滇西地区农村老年人跟骨骨密度与日常生活活动能力(ADL)的关联。方法 采用多阶段随机整群抽样方法抽取滇西4个县(区)22个村中≥60岁的2 287名老年人进行问卷调查、体格检查和跟骨骨密度测量。结果 滇西地区农村老年人ADL的受损率为30.7%。ADL受损者的跟骨超声声速(SOS)和宽带超声衰减(BUA)值均低于ADL未受损者(P<0.001)。多因素Logistic回归分析显示，SOS中水平组和高水平组发生ADL受损的风险分别是低水平组的0.77倍和0.76倍，BUA中水平组和高水平组发生ADL受损的风险均是低水平组的0.77倍。限制性立方样条(RCS)分析显示，SOS、BUA与ADL受损风险呈“U”形关联(P均<0.001)。分层分析显示，女性和60～79岁老年人的SOS、BUA与ADL受损呈负关联(OR<1,P均<0.05),但在男性和≥80岁老年人中无关联(OR包含1,P均> 0.05)。结论 滇西地区农村老年人跟骨骨密度与ADL相关，且SOS、BUA与ADL受损间呈“U”形非线性关联。尤其要关注女性和60～79岁的老年人骨密度状况。