Polymorphism Analysis of MTHFR,Factor II,and Factor V Genes in the Pomeranian Population of Espirito Santo,Brazil

Abstract

Pomeranian populations worldwide immigrated originally from the north of Europe, and because of their preferential marriage, religion, and cultural habits, they show little or no reproductive mixing with local populations. Methylenetetrahydrofolate reductase gene (MTHFR) C677T, Factor V Leiden, and Factor II G20210A polymorphisms are linked to augmented clotting and their frequencies may vary according to population ethnicity. We aimed to assess the frequencies of these thrombophilic alleles in the Pomeranian population residing in Espirito Santo and compare with the general population of the Espirito Santo state, Brazil. A total of 200 individuals were analyzed. The intrapopulation fixation index of the MTHFR C677T polymorphism was 0.03736. The observed heterozygosity was 0.44 and 0.4 for the general and Pomeranian populations, respectively. According to the chi-square test, both populations are in Hardy-Weinberg equilibrium. Four polymorphic alleles were detected for Factor II (2.02%) and 8 for Factor V (4.81%). Our results show that there is gene flow between the general and the Pomeranian population of Espirito Santo, which should no longer be considered an isolated population.

Introduction

The third largest population of Pomeranian descent in the world is found in the mountainous region of the Espirito Santo state, Brazil. This population immigrated originally from the north of Europe, and because of their preferential marriage, religious and other cultural habits, they show little or no reproductive mixing with local populations (Bahia, 2001; Domingues et al., 2006). These habits suggest a considerable degree of endogamy in the Pomeranian population, which indicate a possible founder effect for some genetic conditions.

The methylenetetrahydrofolate reductase gene (MTHFR) is located in the 1p.36.6 chromosomal region, contains 11 exons, and may contain a described thrombophilic polymorphism named C677T (Goyette et al., 1994, 1998). This is a polymorphism present in exon 4, a region of 198 bp (Figueiró-Filho and Oliveira, 2007), and encodes for a transition that converts an alanine into a valine (Frosst et al., 1995), forming heterozygous (CT) and homozygous (TT) variants, which result in enzyme activity reduction (Osian et al., 2007). This genetic variant is related to increased risk for thrombosis, pulmonary embolism, ischemic heart disease, and stroke (Sottilotta et al., 2010). Prothrombin (Factor II) and Factor V gene polymorphisms are also thrombophilic and show similar clinical effects (Stefano et al., 2002). Factor II is encoded by a 20.3 kb gene, located on the short arm of chromosome 11 (Minano et al., 2008). In the 3′ untranslated region of the gene, there is a variant that switches a guanine for an adenine at position 20210 (allele G20210A), increasing plasma concentrations of prothrombin, as well as an augmenting conversion of fibrinogen into fibrin (Junker et al., 1999; Estelles et al., 2000; Asselta et al., 2003; Godoi et al., 2006; Marchiori et al., 2007).

The Factor V gene spans a 100 kb region on the long arm of chromosome 1 (region 23). The Factor V protein is a zymogen with three A domains, one B domain and two C domains. The B domain is displaced from the zymogen after factor V activation, which becomes a cofactor of Factor Xa to convert prothrobin into thrombin. A negative regulation of Factor Va activity is achieved by the proteolytic activity of activated Protein C, which in turn cleaves Factor Va protein at three heavy chain positions (Arg306, Arg506, and Arg679) (Mitterer et al., 1999; Mueller et al., 2005; Medina et al., 2008; Minano et al., 2008). When a guanine at position 1691 is substituted for an adenine (G1691A allele), arginine is changed for glutamine, losing the activated protein C cleavage site, creating a resistance for coagulation inactivation (Nafa et al., 1996).

Because thrombophilic carrier frequencies change according to the population ethnicity and geographical origin (Stefano and Leone, 1995; Tizzano et al., 2002), we decided to study the frequency of thrombophilic polymorphisms (MTHFR C677T, Factor V and Factor II) in the Pomeranian and in the general populations of the Espirito Santo State, Brazil.

Materials and Methods

Sample

Blood samples were collected from individuals of Pomeranian descent (Santa Maria do Jetibá city) and from individuals of the general population of Espirito Santo (Vitoria city, state capital), Brazil. All volunteers signed an informed consent. This study was approved by the Ethics Committee of the Universidade Federal do Espirito Santo (protocol number 053/07). For MTHFR, FII and FV; 100, 99 and 83 individuals from each population were studied respectively.

DNA extraction

Peripheral blood was collected in EDTA Vacutainers or Whatman FTA^® Elute Paper (Whatman, Inc.). Genomic DNA was extracted according to traditional organic extraction protocols or following FTA^® Elute Paper manufacturer's recommendations (Whatman FTA Information, 2005; McClure et al., 2009).

Polymerase chain reaction and restriction fragment length polymorphism

MTHFR, FII, and FV polymorphisms were detected by polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphism (RFLP) analysis, using primers described on Table 1.

Table 1.

PCR Primer Sequences Used to Amplify MTHFR, FII, and FV Genes

Primer	Sequence (5′→3′)
MTHFR	F-TGA AGG AGA AGG TGT CTG CGG GA- R-AGG ACG GTG CGG TGA GAG TG-
FII	F-TCT AGA AAC AGT TGC CTG GC- R-ATA GCA CTG GGA GCA TTG AAC-
FV	F-CAT ACT ACA GTG ACG TGG AC- R-TGT TCT CTT GAA GGA AAT GC-

PCR and cycling conditions are described on Tables 2 and 3. Amplified products were separated in polyacrylamide gels and observed by silver staining. RFLP digestion was achieved using endonucleases HinfI, HindIII, and MnlI for MTHFR, FII, and FV, respectively. PCR products were digested for 12 h at 37°C for HinfI and 35°C for HindIII and MnlI.

Table 2.

PCR Conditions

Primer	Taq pol.	PCR buffer	MgCl₂	Primers	dNTP	DNA	Final vol.
MTHFR	1 u	1X	1.5 mM	0.4 mM	0.2 mM	2.5 μL	25 μL
FII	1.25 u	1X	0.75 mM	0.15 μM	0.1 mM	2.5 μL	25 μL
FV	1.25 u	1X	0.75 mM	0.15 μM	0.1 mM	2.5 μL	25 μL

Table 3.

PCR Cycling Conditions

Primer	Initial denaturation	Denaturation	Annealing	Extension	Final extension	Cycles
MTHFR	94°C, 10′	94°C, 35″	60°C, 35″	72°C, 35″	72°C, 10′	35
FII	94°C, 10′	94°C, 45″	55°C, 45″	72°C, 45″	72°C, 10′	35
FV	94°C, 10′	94°C, 45″	53°C, 45″	72°C, 45″	72°C, 10′	35

A CT transition in the 198 bp MTHFR gene fragment creates a HinfI restriction site, which generates 2 fragments after digestion (175 and 23 bp) (Rahimi et al., 2007). FII (345 bp fragment) restriction with HindIII generates 2 fragments (322 and 23 bp) in thrombophilic alleles. Similarly, MnlI digestion of FV (206 bp fragment) generates 2 fragments (47 and 159 bp) in thrombophilic alleles.

Statistical analysis

Statistical analysis was performed using the Arlequin software, v3.11 (Excoffier et al., 2005).

Results

MTHFR genotypic frequencies for the studied populations are reported on Table 4. The genetic distance coefficient (Fst), a measure of population differentiation based on genetic polymorphisms, was 0.03736.

Table 4.

MTHFR Allele Frequencies in the Studied Populations

	Pomeranian population (n=100)	General population (n=100)
MTHFR C677T
Genotypes n (%)
CC	52 (52.0)	36 (36.0)
CT	40 (40.0)	44 (44.0)
TT	8 (8.0)	20 (20.0)
Alleles n (%)
C	144 (72.0)	116 (58.0)
T	56 (28.0)	84 (42.0)

The observed heterozygote index was 0.44 for the general population, whereas the expected heterozygote index was 0.4872. The chi-square value was 0.9385 and the endogamy coefficient (F) was 1.8211.

For the Pomeranian population, the observed heterozygote index was 0.4, whereas the expected heterozygose index was 0.4032. The chi-square value was 0.0062 and the endogamy coefficient was 1.9921. The chi-square value for the two populations analyzed together was 1.0798.

Four thrombophilic alleles were detected for FII and 8 for FV. Allelic frequencies for FII (G20210A) and FV (G1691A) are described on Table 5.

Table 5.

FII and FV Allele Frequencies in the Pomeranian Population, Espirito Santo, Brazil

Gene	Detected alleles	Carriers	G allele	A allele	A allele frequency
FII	198	4	194	4	2.02%
FV	166	8	158	8	4.81%

MTHFR C6677T polymorphism frequency in different populations worldwide is shown on Table 6.

Table 6.

MTHFR C6677T Polymorphism Frequency in Different Populations Worldwide

	Genotypic distribution [%]			Allelic frequency [%]
Population	CC	CT	TT	C	T
Sao Paulo/Cuiaba	48.3	42.1	9.6	69.4	30.6
Singapore	65.7	32.9	1.4	82.1	17.9
West of Iran	51.7	41.1	7.2	72.2	27.8
India	69.3	30.7	0.0	84.7	15.3
China	32.5	52.8	14.6	58.9	41.1

Discussion

This study analyses three thrombophilic polymorphisms in the Pomeranian and general populations of the Espirito Santo State, Brazil, to verify if preferential marriage has changed the expected distribution of alleles in the two populations.

Our results showed that allelic frequencies were different for the MTHFR gene, suggesting that these are two restricted populations. However, Fst values demonstrate that the genetic distance between the two populations is not statistically significant.

A major effect of endogamy in the genetic composition of a population is the impact it has on the number of heterozygotes. Comparing the expected and observed heterozygosity rates, we detect a smaller number of CT individuals in the general population. Nonetheless, the endogamy coefficient shows an acceptable number for both populations, demonstrating that the general population (F=1.8211) is slightly less endogamous than the Pomeranian population (F=1.9920).

According to chi-square values, both populations are at Hardy-Weinberg equilibrium.

Allelic and genotypic frequencies show that the T allele and the CT and TT genotypes are more frequent in the general population.

The reported frequency of the MTHFR C677T polymorphism worldwide is approximately 35% (Jacques et al., 1996), which is closer to the general Espirito Santo population (20%) than the Pomeranian population (8%). Polymorphism frequency variations due to population ethnicity can be observed in Table 6 (Stefano and Leone, 1995; Tizzano et al., 2002).

This article is the first report of thrombophilic allelic frequencies for a population of Pomeranian descent geographically located in the Espirito Santo State, Brazil. Our results show that there is gene flow between the general and the Pomeranian population of Espirito Santo, which should no longer be considered an isolated population.

Footnotes

Disclosure Statement

No competing financial interests exist.

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