Invest Clin 60(4): 269 - 274, 2019 https://doi.org/10.22209/IC.v60n4a01
Study of the ethnic origin of the prothrombin gene (F2) in the admixed Venezuelan population.
Daniela Kanzler1, Rita Marchi1 and Irene Paradisi2
1Laboratorio Biología del Desarrollo de la Hemostasia, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela.
2 Laboratorio de Genética Humana, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela.
Key words: prothrombin gene (F2); F2 polymorphism 20210 G>A; ethnic origin; rs2070850; rs2070852; rs2282686; rs1799963.
Abstract. The prothrombin (coagulation factor II), codified by the F2 gene, is the precursor of thrombin that cleaves fibrinogen, leading to a blood clot for- mation. The F2 mutation 20210 G>A (c.*97G>A) is associated with prothrom- bin thrombophilia, and carriers have a higher than average risk for developing deep venous thrombosis. The 20210A variant is almost absent in populations other than Caucasoid European, and was not found in a Venezuelan population sample of 160 healthy individuals. To assess the possible ethnic origin of the F2 gene in our admixed population, four intragenic SNPs (rs2070850, rs2070852, rs2282686 and rs1799963), with different allelic frequencies according to eth- nic groups, were studied and compared with the main 1000 Genomes Project super-populations. The results showed intermediate allelic frequencies in all the SNPs, without differentiating a single specific population, confirming the joint ancestral genetic contribution of the parental populations in Latin America: Spaniards, Africans and Amerindians. Our allelic frequency distribution of the F2 polymorphisms was closer to the AMR (American admixed) subset popula- tion of the 1000 Genomes Project. According to this ethnic composition, there is a low probability of detecting carriers of the risk allele 20201A in the Venezu- elan healthy general population.
Origen étnico del gen de la protrombina (F2) en la población venezolana.
Invest Clin 2019; 60 (4): 269-274
Palabras clave: Gen protrombina (F2); polimorfismo F2 20210 G>A; origen étnico; rs2070850; rs2070852; rs2282686; rs1799963.
Resumen. La protrombina (factor de coagulación II), codificada por el gen F2, es un precursor de la trombina, la cual escinde al fibrinógeno, conducente a la formación de un coágulo de sangre. La mutación F2 20210 G>A (c. * 97G>
A) se asocia con trombofilia y los portadores tienen un riesgo superior al pro- medio de desarrollar trombosis venosa profunda. La variante 20210A está casi ausente en poblaciones distintas a la europea caucasoidea, y no se encontró en una muestra de 160 individuos sanos de la población venezolana. Para evaluar el posible origen étnico del gen F2 en nuestra población mezclada, se estudiaron cuatro SNP intragénicos (rs2070850, rs2070852, rs2282686 y rs1799963) con diferentes frecuencias alélicas, según los grupos étnicos y se compararon con las principales superpoblaciones del Proyecto 1000 Genomas. Los resultados mostraron frecuencias alélicas intermedias en todos los SNP, sin diferenciar una única población específica, lo que confirma la contribución genética ancestral conjunta de las poblaciones parentales en América Latina: españoles, africanos y amerindios. Nuestra distribución de frecuencias alélicas de los polimorfismos de F2 fue más parecida al subconjunto AMR (American admixed population) del Proyecto 1000 Genomas. De acuerdo con esta composición étnica, existe una baja probabilidad de detectar portadores del alelo de riesgo 20201A en la población general sana venezolana.
Received: 13-09-2018 Accepted: 09-10-2019
Prothrombin is a proenzyme that is converted to thrombin by the prothrom- binase complex (Factor Xa and Factor Va, assembled on a phospholipidic surface in the presence of Ca+2). Thrombin cleaves fibrinogen, which triggers a self-aggrega- tion process that end with clot formation. Approximately, 49% of prothrombin levels seem to be genetically regulated (1). The prothrombin polymorphisms 20210 G>A (c.*97G>A, rs1799963) (2) and 19911 A>G (c.1726-59 A>G, rs3136516) (3) have
been associated to increased prothrombin plasma levels, and both represent indepen- dent risk factors for venous thrombosis (4-6). The 20210A genetic variant has dif- ferent prevalence according to the ethnic group, being more frequent in Caucasians, especially Spaniards and Southern Europe- ans (French, Italians, Greeks) (7-12). We have assessed the prothrombin polymor- phism G20210A in a Venezuelan population sample, recruiting 160 healthy subjects from the metropolitan area of Caracas, as described elsewhere (13). All of them were homozygous for the ancestral allele G.
The allele A is a worldwide infrequent variant, which is absent in Asian and Afri- can populations, and is at very low frequen- cies in some European populations: Azorian (0.018), Basque (0.020), Spaniards (0.015),
Cypriot (0.039), French (0.021) (ALFRED database, https://alfred.med.yale.edu), with a global Minor Allele Frequency (MAF) of 0.0036 (1000 Genomes Project, http://www. internationalgenome.org). In the American subset populations (AMR) included in the 1000 Genomes Project (Mexican, Colombi- an, Peruvian and Dominicans), the allele A frequency is 0.0144, similar to those found in the above mentioned populations. The ab- sence/low frequency of the allele A in non- Caucasian populations have been attributed to an ancient founder effect, occurred after the divergence of Africans from non-Africans (14). On the other hand, its frequency was found between two to ten-fold increased in patients with idiopathic vein thrombosis compared to healthy control subjects (2). Thus, the absence of the allele A in this Ven- ezuelan cohort prompted us to assess the probable F2 gene ethnic origin in the ad- mixed Venezuelan populations.
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A cohort of 160 healthy subjects (aged between 25 to 60 years old) from the metro- politan area of Caracas was selected. A 5 mL blood sample was withdrawn in 15% EDTA- Na , and DNA was extracted as described elsewhere (13). The project was approved by the Bioethical Committee of the Venezuelan Institute for Scientific Research (IVIC). A signed informed consent was obtained from all participants.
Prothrombin polymorphism G20210A. Genotyping of the G20210A polymor- phism (rs1799963) was performed by DNA PCR amplification followed by Hind III re- striction endonuclease digestion, as previ-
ously described (13).
F2 intragenic polymorphisms
Three intragenic prothrombin (F2) polymorphisms: c.240+83 C>T (IVS 2; rs2070850), c.423-7 G>C (IVS 5; rs2070852) and c.1472+251 T>C (IVS 11; rs2282686),
which have clearly different allelic frequen- cies in distinct ethnic groups were selected. Primers were designed with Primer-BLAST software (at https://www.ncbi.nlm.nih.gov/ tools/primer-blast/) and each SNP was de- tected by restriction analysis, as follow:
rs2070850 (C>T): (f) 5’-GAGAGTGC- GTGGAGGAGAC and (r) 5’-CATGTCATG-
GAGCTGCACA, product size 286 bp. Allele T produces a recognition site for BsmI re- striction enzyme.
rs2070852 (G>C): (f) 5’-CCAC- CATGGGCTGAGAAC and (r) 5’-CATTCCT-
GCCTCCTCACG, product size 195 bp. Allele C is recognized by the restriction endonucle- ase MnlI.
rs2282686 (T>C): (f) 5’- AAGAGCCCC TTTCCCTTTTC and (r) 5’- GGTGAAACCCA
CCAGTCTCT, product size 266 bp. Allele T abolishes the restriction site for SmaI en- zyme.
Allelic frequencies were calculated by direct counting. Hardy-Weinberg equilibrium was assessed for each SNP, using the χ2 test.
All the ancestors (up to two genera- tions back) of the individuals recruited in the present study were born in Venezuela. The 160 individuals studied were homozy- gous for the wild type allele G of the pro- thrombin polymorphism G20210A.
The allele frequencies for the three SNPs are summarized in Table I. All of them were in Hardy-Weinberg equilibrium.
The allelic frequencies obtained for each polymorphism was compared to the five super-populations reported in the 1000 Ge- nomes Project: Admixed American (AMR), European (EUR), African (AFR), South Asian (SAS) and East Asian (EAS) (Table I).
ALLELIC FREQUENCIES OF THE THREE INTRAGENIC PROTHROMBIN (F2) POLYMORPHISMS IN DIFFERENT POPULATIONS.
Populations
rs2070850 C>T
Ancestral allele: C
rs2070852 G>C
Ancestral allele: G
rs2282686 T>C
Ancestral allele: C
VZL | C=0.780 T=0.220 | G=0.439 C=0.561 | T=0.406 C=0.594 |
AMR | C= 0.718 T=0.282 | G=0.416 C=0.584 | T=0.590 C=0.409 |
EUR | C=0.879 T=0.121 | G=0.292 C=0.708 | T=0.709 C=0.290 |
AFR | C=0.970 T=0.030 | G=0.873 C=0.126 | T=0.210 C=0.790 |
SAS | C=0.830 T=0.170 | G=0.293 C=0.706 | T=0.707 C=0.292 |
EAS | C=0.438 T=0.562 | G=0.697 C=0.303 | T=0.301 C=0.698 |
VZL: Venezuelan (present study); AMR: American; EUR: European; AFR: African; SAS: South Asian; EAS: East Asian. Number of chromosomes in the Venezuelan sample: rs2070850 (n= 304); rs2070852 (n= 312); rs2282686 (n=320).
Two of the three studied polymorphisms had intermediate frequencies for each allele, different from that of the main parental pop- ulations (European, African and Asian).
The prothrombin 20210G>A transi- tion (c.*97G>A, rs1799963), despite being located in the 3’ untranslated region of the gene, has been associated with elevated plas- ma prothrombin levels that raises the risk of thrombotic events. The pathophysiologic mechanism of this genetic variation seems to be a gain-of-function mutation that pro- duces an enhanced 3’ end processing and in- creased mRNA accumulation, with the con- sequent increase in protein synthesis (15).
First reports of allelic frequencies showed that 18% of probands from thrombo- philic families and 6% of nonrelated patients with deep vein thrombosis carried the allele A, contrasting with the lower frequencies in healthy controls (between 1 to 2%) (2, 16).
Subsequent additional studies found that the allele A is almost absent in non Cauca- soids populations. In the Venezuelan sample of 320 chromosomes of healthy individuals its frequency was null, even though at least one carrier is expected. This motivated us to assess the probable ethnic origin of the F2 gene, using population-specific intragenic polymorphic markers.
Venezuelan populations are the result of the genetic admixture of African, Euro- pean and Amerindian ethnic groups (17). We chose three intragenic prothrombin (F2) polymorphisms: c.240+83 C>T (IVS 2; rs2070850), 423-7 G>C (IVS 5; rs2070852) and c.1472+251 T>C (IVS 11; rs2282686),
which have clearly different allelic frequen- cies between distinct ethnic groups.
It was not observed an allelic disequi- librium in the F2 gene polymorphisms stud- ied of the F2 gene that allows to propose a greater similarity with a particular ethnic group, as have been reported for other pop- ulations (Table I), where one of the alleles
have a higher frequency compared to other super-population samples.
The AMR population subgroup of 1000 Genomes Project was the exception, which showed greater similarity with the Venezu- elan population. The individuals included in the AMR group belong to American ad- mixed populations with the same parental origins as Venezuelan population (Spanish, Amerindian and African). Thus, the inter- mediate allelic frequencies observed in Ven- ezuelans confirmed the mixed ethnic origin of our population, and the similar genetic background between the Ibero-American populations. The absence of 20210A allele in the 320 chromosomes examined suggests that in this cohort, the Caucasian/Spanish contributions must be low, besides being a sample of healthy individuals, in whom the A allele should be less frequent.
In conclusion, a unique, discernible and/or probable ethnic origin of the F2 gene in the very heterogeneous healthy population of the metropolitan area of Caracas (Capi- tal District) cannot be established with the intragenic polymorphisms used, although a clearly admixed pattern of the population was supported.
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