Introduction

Several studies that have been carried out on diverse human populations to analyze the molecular bases of deficiencies of the blood clotting Factor XIII have led to the identification of five frequent polymorphisms located on the exonic regions of the gene which encodes for the A subunit of the plasmatic Factor XIIIA. One of those polymorphisms is the SNP (ie, single nucleotide polymorphism) Val34Leu, consisting in the substitution of guanine by thymine on exon 2 of the gene encoding this subunit, which in turn changes the aminoacid Valine by Leucine in the position 34 of the protein, three amino acids away from the site of activation by thrombin ₁,₂,₃ . FXIIIA Val34Leu is a frequent polymorphism that has been related to higher levels of transglutaminase activity; while the biochemical consequences are not yet well understood, it has been reported that activation of FXIIIA by thrombin occurs 2 to 3 times faster in the presence of the Leu34 variant, seemingly by alterations of fibrin cross linking. In some populations this molecular variant shows a relationship with the occurrence of thrombotic and hemorrhagic processes, conferring protection against myocardial infarction and venous thromboembolism, and predisposing to intracranial hemorrhage ₄,₅,₆,₇,₈ . Also, various studies performed on diverse human groups have revealed a wide variability for this polymorphism. Available reports indicate that the Leu34 allele does not predominate in any of the tested populations but it shows noticeable variations in frequency, with high values in Amerindians, followed by Europeans and lower values in Africans and almost nil in Asians ₁,₉,₁₀ . Based on such findings some authors agree that this polymorphism shows a frequency level related to ethnic groups but it does not represent an important genetic marker of populations. Little work has been done on American hybrid or urban populations and data are restricted to the US and Brazil ₅,₉,₁₁,₁₂ . Both countries emerged from the confluence of European, Amerindian and African population groups which were differentially involved in the colonization process which in turn led to a great genetic heterogeneity.

Preceding studies have pointed out the importance of adequate sample selection to evaluate frequency distributions of various polymorphisms in apparently healthy populations, prior to analyses of relationships with any kind of pathology. Particularly, with high proportions of racial admixture, mistakes of interpretation may be inherent to the process of genetic stratification of such populations ₁₃,₁₄,₁₅,₁₆ . For this reason and in order to minimize such errors, some researchers have preferred to restrict their samples to individuals from the same ethnic group ₈ . The geographic distribution of the Leu34 variant may explain at least in part, the expressions of blood-clotting disorders in different population groups ₉,₁₀ ; although despite the high relevance of Val34Leu in epidemiological studies, little is known regarding its variations in populations from similar origins ₁₇ . The aim of this study was to elucidate the genetic differentiation patterns of the Leu34 allele, analyzing its frequency distribution as reported for various worldwide populations. We also publish the frequencies of this polymorphism in apparently healthy urban people from three States of Eastern Venezuela, trying to correlate these values with their levels of racial admixture.

Populations And Methods

Populations

Methods

Statistical analyses

Results And Discussion

Most studies dealing with Val34Leu frequencies have been done on European populations but little attention has been paid to regional and ethnic features of the individuals comprising the studied samples, thus narrowing the scope for broad conclusions. Review of the frequency distributions of the five ethnic/geographic groups chosen for the present analysis (Table 1) confirms that the highest values for the Leu34 allele correspond to native American people, with an average of 29.3%, followed by Europeans (25%) and Africans (18.1%) and the almost inexistence of this allele in Asians (0.6%). These values reveal an important pattern of genetic variation among main genetic/geographic groups, where the only allele frequencies not significantly different between each other were those between Europeans and Amerindians (p=0.1), in accordance with a previous study by Attié-Castro et al. ₉ .

Figure 1

Table 1: Frequency of Val34Leu in world-wide populations

Livshits and Nei ₂₁ propose estimates Dm and Gst` to compare the extent of genetic differentiation for various groups of populations. In the present analysis, the values of Dm and Gst` between four continental groups compared are, 0.0319 and 10.41%, respectively (Table 2), showing that genetic diversity and differentiation between these groups is higher than that observed for intra-continental/geographic comparisons. This result resembles those reported for some STR loci ₂₇ and confirms an ethnic heterogeneity in the prevalence of the Leu34 allele.

Figure 2

Table 2: Interpopulation gene differentiation coefficients (Gst` and Dm) within and between genetic/geographic groups.

A noticeable genetic diversity can also be detected within each continental group; native Americans, in addition to high frequency values, show marked differences between populations and these are reflected by the high values of Dm (0.0141) and Gst` (3.35%), as has been previously pointed out for other polymorphisms, attributed to pronounced evolutionary and populational events such as genetic drift ₂₈,₂₉ .

From Tables 1 and 2 it emerges that Europeans show an interesting pattern of Val34Leu frequency distributions, with an intermediate diversity value (Gst`=1.74%) which appears to be caused by minor regional differences. On that continent the highest values of Leu34 occur in the central-eastern (28.0%) and northern (25.0%) portions and the lowest in the southern ones (23.3%). At first glance, the most homogeneous region is the south (Gst`=0.8%), but the greater heterogeneity of the central-east diminishes considerably from 3.16 to 0.3% when Germans are excluded from the group, since they show an extremely low frequency of Leu34 (11%) in a rather small population sample (N=14). Excluding this country, the greatest European heterogeneity appears in the northern region, due to the data reported for Finland ₂,₃₀ where genetic drift events have been widely documented ₃₁ . It may be stated that Europeans do not always show high Leu34 frequencies as has been claimed, as those from the southern or Mediterranean regions have lower values, analogous to those reported for African groups. The scarcity of data pertaining Africans and Asians does not allow any conclusive arguments; however, it is clear that reported frequencies are low in the case of Africans, even though with a high heterogeneity (Gst`=2.62%), and the very low frequencies of the Leu34 allele in Asian groups studied so far is reflected by a low value of internal diversity (Gst`= 0.6%).

It is not clear what biological advantage could be conferred to carriers of allele Leu34 that allows it to remain instead of becoming deleted by the process of selection. The high frequency of this allele in American natives is noteworthy, in contrast with the extremely low Asian values. It may be suggested that its frequencies in other Asian populations might be greater or alternatively, that the observed values support the influence of extreme events of genetic drift on Amerindian people, as proposed earlier by ₂₈,₂₉ . Such considerations have led to suggest that it is an old mutation which appeared in humans prior to the African/non-African racial divergence ₉ , with subsequent variations throughout continents due to complex population and evolutionary processes. The groups belonging to the American admixed populations deserve special attention, as they have a shorter genetic history as the product of genetic exchanges between the other four groups thus far considered. The few available reports concern populations from the US and from Brazil, plus the data published here for Venezuela, with an overall wide range of allele Leu34 frequencies together with an intermediate coefficient of diversity Gst` of 1,97%. The kind of racial admixture experienced by those populations explains in part their frequency distribution. Thus, the highest Leu34 values correspond to whites from Washington (26%), analogous to their northern-European ancestors ₇ . The data published by Attié-Castro et al. ₉ for Brazilians of European extraction, most of them living in Riberão Preto, in the State of São Paulo, reveal higher frequencies (28.3%) than in their Portuguese ancestors; it might be inferred that an appreciable Amerindian contribution could have increased the Leu34 frequency in this population sample. In black North Americans and Brazilians the observed allele frequencies resemble the few available data known for their African ancestors, 19.5 and 14%, respectively ₉ .

Our results for the three Venezuelan populations do not show significant differences between them, although the Leu34 frequency is higher in Monagas (23%) than in the States of Sucre (18%) or Nueva Esparta (17%). These last two values are among the lowest reported for American admixed people, even though these populations have a predominant European racial contribution, that is, 84.6% in Sucre and 79.8% in Nueva Esparta (Table 3); however, that European contribution came from southern Spain, in which the Leu34 prevalence is rather low. The higher frequency found in Monagas may be attributed to the greater Amerindian component (23%) noted in this State in the admixture analysis, as compared to those obtained in Sucre and Nueva Esparta, 6% and 5%, respectively.

Figure 3

Table 3: Gene frequencies for ABO and RH systems and admixture estimates in the three Venezuelan populations studied.

This report represents the first approach for a populational study of the Val34Leu polymorphism. Previous data on frequencies for allele Leu34 came from healthy control groups in epidemiologic genetic studies, and this condition might produce some bias on allele frequency estimation. In our analysis, we used a wider number of European populations than those used in previous reports, and we evaluated the population diversity of allele frequencies with interpopulation gene diversity Dm and population genetic differentiation index (Gst`), which gives a clearer picture of the Leu34 distribution among Caucasoids.

In recent years various researchers have agreed to use the terminology “population genetic stratification” to refer to genetic features resulting in populations which are continuously subjected to racial mixing, or in those which have become mixed more recently, that is, those whose genetic structure has been modified only a few generations back ₁₆,₃₂,₃₃ . In this sense, the current populations of the American continent show a wide heterogeneity, both between and within them, which translates into a broad genetic stratification; in some circumstances this could lead to wrongly associate some loci with diseases or other disorders ₃₄ . Hence, in such association studies care should be taken to avoid false interpretations ₃₅,₃₆ . The Leu34 allele cannot elude this heterogeneity effect in its geographic and ethnic distribution in the Americas, as reflected by its interpopulation diversity coefficient, Gst`=1.97. The ethnic heterogeneity of the distributions of some related polymorphisms, besides that of Val34Leu, has already been documented by various studies of cardiovascular diseases ₃₇,₃₈ . Hence, it becomes important to elucidate the degree of variability of the genes involved to understand the diverse patterns of pathologies of the haemostatic system.

To conclude, the overall results indicate that whereas Val34Leu does not yield conclusive information regarding genetic affinities, however reveals differences between human groups with differing genetic origins and its frequency in mixed American populations varies in relation to their parental contributions. This polymorphism is associated with both thrombotic and bleeding pathologies and its functions ought to be assessed together with other polymorphisms associated to homeostasis, as well as their geographic and population heterogeneities. This should help to diminish any biases brought about by genetic stratification effects on populations with heavy proportions of racial admixture.

Acknowledgements

Correspondence to

Dinorah Castro de Guerra, M.Sc, Dr, Laboratorio de Genética Humana, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC), Apdo. 20632, Caracas 1020A, Venezuela. Tel. +582125041087; Fax +582125041086; E-mail: dcastro@ivic.ve