Correlation Between Single Nucleotide Polymorphism of Prothrombin Gene G20210 and Deep Vein Thrombosis after Total Joint Replacement in Chinese Patients

Abstract

Object: To evaluate the correlation between prothrombin gene G20210A mutation and deep vein thrombosis after total joint replacement through detecting distribution frequency of single nucleotide polymorphism of the gene in patients undergoing total joint replacement. Method: PCR and direct sequencing of DNA are used to analyze the frequency of prothrombin gene G20210A mutation in 55 patients undergoing total joint replacement, and the relationship between the mutation and deep vein thrombosis after total joint replacement is evaluated. Result: Morbidity of prothrombin gene G20210A mutation in 55 patients is 0, both in the DVT group and non-DVT group. There is no significant difference between the two groups (p > 0.05). Conclusion: Mutation of prothrombin gene G20210A in Chinese patients is rare, and there is no correlation between the genetic mutation and deep vein thrombosis after total joint replacement.

• prothrombin gene
• deep vein thrombosis
• total joint replacement
• Chinese

INTRODUCTION

Recently, foreign studies have shown that polymorphism of untranslated region G20210A at the 3'end of prothrombin gene is associated with venous thromboembolism (VTE), and mutation of the gene is a risk factor of VTE after total joint replacement (TJR). Some domestic researchers have found no correlation between the gene and VTE in Chinese patients, but no paper about the relationship between the gene mutation and DVT after TJR has been published yet. In our study, PCR and direct sequencing techniques are used to detect the frequency of prothrombin gene G20210A mutation, and the relationship between the mutation and deep vein thrombosis after total joint replacement in Chinese patients is evaluated.

MATERIALS AND METHODS

Materials

55 Chinese patients, who underwent total knee or hip replacement and were given low molecular weighed heparin (LMWH) prophylactically during the period of April to August of 2004, were selected. 4 of them combined with DVT of lower limbs preoperatively. 5 millimeters of peripheral complete blood drawn from each patient was preserved under the condition of −80°C after being anti-coagulated with sodium citrate.

Methods

• Extract and purify the DNA rapidly.

• Amplification of prothrombin gene: primers that contain the G20210 position of prothrombin gene are designed according to the papers of Poort [1] and Ripol [12] et al. The sequence of primers are as following:

• Upriver primer: 5’-TCTAGAAACAGTTGCCTGGC-3’

• Downriver primer: 5’-AGGTGGTGGATTCTTAAGTC-3’

• PCR productions are identified by agarose gel electorphoresis.

• Amplified fragments of prothrombin gene G20210 are sequenced directly.

Statistic methods

SPSS 11.0 statistic software is used for database setup and analysis. Measurement data are shown in the form of mean±standard deviation. T-test is used to testify mean differentia between groups. If data is not normally distributed, Mann-Whitney U-test will be selected for further statistic analysis. Ratio or rate is used for counting data. Genetic counting process is applied for genetic frequency calculation. χ²-test is used to determine whether the genotype distribution agrees with the Hardy-Weinberg hereditary balancing law, and differentia of allelic gene frequence between groups as well. The logistic regression is applied for multi-factor analysis. P < 0.05 represents that the differentia is statistically significant.

RESULTS

A target DNA fragment of 419 bps is amplified for each patient (Figure 1).

Figure 1.  Electrophoresis result of Prothrombin gene G20210 DNA amplification fragment in TJR patients. M: DNA Marker 1–10 patients number.

Successful sequencing has been performed for all the fragments of the 55 patients. No case of mutation of prothrombin gene G20210A has been discovered (Table 1). A female client whose number is 39 suffered DVT after bilateral total knee replacement. A G-to-T mutation was found on the 20208th position instead of G-to-A mutation on the 20210th position (Figure 2).

Figure 2.  Sequencing result of patient 39 who suffered DVT postoperatively. There was not a G-to-A mutation in the 20210^th position (276^th in the figure), but a G-to-T mutation in the 20208^th position (274^th in the figure) was found. AT of the patient was 117% preoperatively, and 89.8% postoperatively.

Table 1. Sequencing results of prothrombin gene G20210A of all patients

No.	Gender	Age (years old)	Surgery	Side	DVT	Mutation of prothrombin G20210A
1	F	57	TKR	Uni	yes	no
2	M	67	THR Revision	Uni	no	no
3	M	47	THR	Bi	no	no
4	M	37	THR	Uni	no	no
5	F	71	THR	Uni	yes	no
6	F	60	TKR	Bi	yes	no
7	F	66	TKR	Bi	yes	no
8	F	63	TKR	Uni	yes	no
9	F	76	THR	Uni	no	no
10	F	57	TKR	Bi	yes	no
11	F	67	TKR	Bi	Yes, proximal	no
12	F	70	TKR	Bi	no	no
13	F	66	TKR	Uni	yes	no
14	M	67	THR	Uni	no	no
15	F	72	THR	Uni	no	no
16	M	47	THR	Uni	no	no
17	F	51	THR	Uni	no	no
18	M	60	THR Revision	Uni	no	no
19	F	51	THR	Uni	no	no
20	M	45	THR	Bi	no	no
21	F	58	THR	Uni	Yes, previous	no
22	F	33	THR	Uni	no	no
23	F	59	THR	Uni	yes	no
24	M	76	TKR	Uni	no	no
25	F	74	TKR	Uni	yes	no
26	F	27	TKR	Uni	no	no
27	F	74	TKR	Uni	yes	no
28	F	23	THR	Uni	no	no
29	F	57	THR	Uni	yes	no
30	F	72	TKR	Bi	yes	no
31	F	47	THR	Bi	yes	no
32	F	58	TKR	Bi	yes	no
33	F	49	TKR	Uni	no	no
34	F	51	TKR	Uni	yes	no
35	F	68	TKR	Uni	Yes, previous	no
36	M	70	TKR	Uni	yes	no
37	M	40	THR	Bi	no	no
38	F	72	THR	Uni	no	no
39	F	60	TKR	Bi	yes	no*
40	F	66	TKR	Bi	Yes, previous	no
41	M	65	THR	Uni	no	no
42	F	43	THR	Uni	no	no
43	F	58	TKR	Uni	yes	no
44	F	56	THR	Uni	no	no
45	F	67	THR	Uni	no	no
46	F	70	TKR	Uni	yes	no
47	F	52	THR	Uni	no	no
48	F	64	TKR	Uni	yes	no
49	F	63	THR	Bi	yes	no
50	F	76	TKR	Bi	Yes, previous	no
51	M	75	TKR	Bi	no	no
52	F	60	THR	Uni	no	no
53	M	28	THR	Uni	no	no
54	F	66	TKR	Uni	Yes, proximal	no
55	F	50	THR	Uni	no	No

“previous” in the “DVT” column refers to preoperative DVT of lower limb(s) of the patient;

“*” refers to some other mutation of prothrombin gene at G20210 position, detailed in Figure 1.

27 of the 55 patients combined with DVT after TJR, while the other 28 did not. Differentia of age, body mass index (BMI), preoperative prothrombin activity, gender ratio, and type of surgery between the two groups were significant (p < 0.05) (Table 2).

Table 2. Statistic comparison of DVT group and non-DVT group after TJR

Item	DVT Group (n = 27	Non-DVT Group (n = 28)	t	x2	P
Age (years old)	63.67±7.14	53.18±16.58	3.066		0.004*
BMI (kg/ m2)	27.67±3.60	23.65±3.32	4.311		0.000*
Preoperative AT (%)	108.10±21.35	94.76±13.96	2.732		0.009*
Gender M	1 (37%)	12 (42.9%)
F	26 (96.3%)	16 (57.1%)		11.674	0.001*
Surgery TKR	21 (77.8%)	5 (17.9%)
THR	6 (22.2%)	23 (82.1%)		19.8	0.000*

*differentia between groups is significant.

A sample of prothrombin gene G20210A mutation was not detected in our series. Genetic frequencies of both heterozygote and mutational homozygote are 0. The variation frequency of prothrombin G20210A genotype is 0, in either DVT or non-DVT group, and the mutation frequency of each group is 0 as well. The appearance of G20210 allele is 0, and there is no significant difference between the two groups (p > 0.05) (Tables 3 and 4).

Table 3. Genotype comparison of DVT group and non-DVT group after TJR

Genotype	DVT group (n = 27)		Non-DVT group (n = 28)
	Frequencies	Percentage	Frequencies	Percentage
Wild homozygote GG	27	100	28	100
Heterozygote GA	0	0	0	0
Mutational homozygote AA	0	0	0	0

Table 4. Allelic comparison of DVT group and non-DVT group after TJR

Allele	DVT group (n = 27)		Non-DVT group (n = 28)
	Frequencies	Percentage	Frequencies	Percentage
G	27	100	28	100
A	0	0	0	0

DISCUSSION

Thrombin plays the role of a hinge, which can promote the process of transition from fibrinogen to fibrin 3–6.

In 1996, Poort et al. [7] discovered that G-to-A mutation of untranslated region G20210 at the 3'end of prothrombin gene is associated with the elevation of plasma prothrombin level, which increases the risk of VTE. Numerous studies have been performed, and frequency in different populations has been reported. It is thought that the variation of G20210A is an important risk factor of embolism in the European population, but the variation is rarely reported in Chinese patients. Foreign studies have discovered that mutation of G20210A is one of the risk factors of symptomatic VTE after TJR, but there is not a domestic series on this topic.

PCR and direct sequencing technique have been used to detect the G-to-A mutation of the 20210^th position of prothrombin gene in 55 patients who underwent total joint replacement surgery. According to the literature [7], [16], we conformed the primers designed by the two scholars, and designed our own primers as follows: upriver prime: 5’-TCTAGAAACAGTTGCCTGGC-3’ and downriver primer: 5’-AGGTGGTGGATTCTTAAGTC-3’. Comparing with the DNA sequence, we can amplify a 419bp fragment. Of the amplified fragment whose sequence is 5’-… AGCGAGCTTCAA…-3’, the 20210th nucleotide (underlined G) is about 100 bps away from the 3’ end, which can increase the accuracy of sequencing.

MOLECULAR STRUCTURE AND FUNCTION OF PROTHROMBIN

Prothrombin is a kind of single-chain glycoprotein synthesized in liver, whose molecular mass is 72kd, and contains 579 amino acid residues. It is made up with some functional regions: N-end is a γ-hydroxyl glutamic acid region, and C-end is the catalytic region of prothrombin, where thrombin, the activated form of prothrombin, is formed. Region between the two above is the Kringle structure. Under the activation of prothrombin complex, the prothrombin transforms to thrombin. Thrombin further activates coagulation factor V, VIII, XII, and activates a series of positive feedback reactions to transform fibrinogen to fibrin and coagulate the blood. It can activate protein-C system as well, which will promote anti-coagulation activity and gather activated platelets 4–6. Of the 55 patients, preoperative prothrombin activity in patients of the DVT group is significantly higher than of the non-DVT group (box-2), which means high activity of prothrombin is a risk factor of postoperative DVT.

RELATION BETWEEN POLYMORPHISM AND FUNCTION OF PROTHROMBIN

Protein structure change due to genetic mutation is one of the important factors of embolism during the process of coagulation and fibrolysis [16]. The position of the prothrombin gene on human chromosome is 11p11-12. Total length of the gene is 21kb. Position of G20210 is at or near the split point of the untranslated region of the 3’ end of mRNA. G20210A mutation can result in the intruding of nucleotide at or near the point, and lead to alternation of genetic sequence. The change brings higher translation efficiency of the gene or higher stability of transcript mRNA, and leads to the elevation of plasma prothrombin level. Research on hereditary thrombophilia has discovered that the 20210 G-to-A mutation of the 3’ untranslated region increases the risk of VTE by 3 times. In spite of age or gender, risk of VTE elevates in the population with 20210A allele. With higher activity of prothrombin preoperatively, none of the 55 patients in our series has reported the mutation, which may suggest that the elevation of prothrombin activity has nothing to do with the genetic mutation.

DISTRIBUTION FREQUENCY OF PROTHROMBIN GENE MUTATION AND VTE

There are regional and racial differences of prothrombin gene mutation between populations. Generally, some certain mutation exists in Europeans, but the frequency can be very low or absent in African-American or Asian populations [1], 17–22, 7–11. Two domestic studies [24], [25] have found only 4 carriers of G20210A gene in normal population, all of whom are heterozygous, and not a G20210A homozygous or heterozygous mutation has been reported in other studies 10–13.

Significant differences of the genetic mutation exists among different races. Risk of thromboembolism in the West is significantly higher with the G20210A mutation [1], 17–22, 7–11. A case-controlled study based on populations has demonstrated that the 20210A allele is an ordinary allele, which can increase the risk of VTE by 3 times [7]. All studies have proved that the 20210 G-to-A mutation is a separate risk factor of venous thromboembolism.

In China, Bi et al. have analyzed the polymorphism of prothrombin gene G20210 and reported a significantly higher mutation frequency in the DVT group, but a sample of mutation was not reported in any other studies. It is thought that the polymorphism of prothrombin gene G20210A is not a risk factor of VTE in Chinese patients. In our series, we have not detected the mutation in the 55 patients, of which the result agreed with most domestic studies. One patient, whose preoperative prothrombin activity was 117%, had a 20208 G-to-T mutation detected (Figure 1). More research is necessary to explain the relationship between the G20208T mutation and VTE.

RELATIONSHIP BETWEEN THE MUTATION OF PROTHROMBIN GENE AND DVT AFTER TJR

The mutation of prothrombin gene G20210A is reported as a risk factor of symptomatic VTE after TJR in foreign studies [14], [15], but there is not a similar report domestically.

PCR and direct sequencing techniques were used to detect the frequency of prothrombin gene G20210A mutation for the first time; a case of mutation was not detected. Although DVT after TJR is strongly correlated with elevated preoperative prothrombin activity, 0 of prothrombin gene mutation demonstrates that:

1. Mutation of prothrombin gene G20210A is rare in the Chinese population, and is not associated with DVT after total joint replacement.

2. Elevation of plasma prothrombin activity in high-risk groups of DVT after TJR is not associated with the mutation of prothrombin gene G20210A, and there may be other polymorphism of the gene or some other risk factors.

3. Differences of age, body mass index (BMI), preoperative prothrombin activity, gender ratio, and type of surgery between the two groups being significant, DVT after TJR in our series can be related with any of these factors.

CONCLUSION

Mutation of prothrombin gene G20210A is rare in the Chinese population, and it is not correlated with DVT after TJR. Elevation of plasma prothrombin activity in high-risk groups of DVT after TJR is not associated with the mutation of prothrombin gene G20210A. There may be other polymorphism of prothrombin gene or some other mechanism.

Acknowledgement

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.