Genetic risk factors for venous thromboembolism among infertile men with Klinefelter syndrome.

BACKGROUND: Klinefelter syndrome (KS) is one of the commonest sex chromosome disorders. Affected males become infertile and highly susceptible to several health problems, including vascular thromboembolism (VTE). The risk of VTE may be exacerbated by an underlying genetically inherited thrombophilia. In this study, we aimed to investigate the genotype and allele frequencies of common gene polymorphisms related to hereditary thrombophilia in infertile males with KS compared to normal, fertile men.
METHODS: Eighty-five infertile males with KS and 75 healthy control males were included in this case-control study. Genetic testing was done using an extended thrombophilia gene panel by Multiplex PCR reverse hybridization method.
RESULTS: There was an increased frequency of mutant alleles and heterozygous genotypes of FV Leiden, FV H 1299R, Pro G20210A, MTHFR C677T and PAI-1 4G/5G thrombophilic gene polymorphisms in KS patients compared to the control group. It was shown that 10.7% of KS patients had the A3 haplotype of the EPCR gene in comparison to 5.3% of control patients. The A3/A3 genotype was found only in KS patients (7.1%). Carriers of more than one mutant allele in KS patients exceeded the control (p < 0.001).
CONCLUSION: A high prevalence of thrombophilic gene polymorphisms and the coexistence of different mutant alleles were evident in infertile KS males. These data highlight the importance of conducting further studies to understand the role of hereditary thrombophilia in predicting venous thrombosis in patients with Klinefelter syndrome.

Introduction

In 1942, Dr. H. Klinefelter et al. first described a set of symptoms that has come to be known as Klinefelter syndrome (KS). With an incidence of about 1 in 500–1000 newborns, it is the most frequently occurring sex chromosome disorder in males [1], [2]. Klinefelter syndrome is the most common cause of male infertility and hypogonadism and represents almost11% of all cases with azoospermia and 0.7% of all cases with oligozoospermia [3].

KS is the commonest sex-chromosome disorder caused by the nondisjunction of chromosome X and results in either a classic form, in which there is an extra copy of chromosome X (47, XXY), or a mosaic form [4], [5].

Both morbidity and mortality are significantly increased in KS, This may be explained by the fact that KS patients are at an increased risk of developing serious medical problems that include the following: 1-Motor, cognitive, and behavioral dysfunction, 2-Tumors and cancer, 3-Vascular diseases, 4-Endocrinal, metabolic, and autoimmune diseases [6]. Other mechanisms could be attributed to altered gene dosage due to the presence of an extra X chromosome, or disturbed inactivation of some genes carried on the X chromosome [7], [8]. Endocrinal disturbances and delayed testosterone treatment are also recognized as aggravating factors [9], [10].

These observations were supported by epidemiological data from Denmark and Britain that reported a significant increase in morbidity and mortality rates (70% and 50% respectively) in males with KS relative to controls [7], [8]. Venous thromboembolism (VTE) has been described in a number of case reports and the morbidity and mortality rates caused by VTE in men with KS were highlighted by Bojesen et al. in 2006 through a large study in Denmark that was conducted on 832 KS patients and 4033 controls. They found that 39 KS patients had deep vein thrombosis and 19 KS patients had pulmonary embolism [8].

Furthermore, an epidemiological Swedish study reported a high relative risk of VTE in 1085 KS males compared to the normal male population. The standardized incidence ratio was 12.10 (95% CI 6.22 to 21.21) in those <30 years old, and 11.00 (95% CI 7.86 to 14.99) in those aged30- 49 years. They concluded that KS men are more prone to the risk of thromboembolism than the general population [11].

Hereditary thrombophilia is a precipitating factor of VTE. It is well established that hereditary thrombophilia is associated with mutations in several genetic loci related to coagulation and fibrinolysis cascades. In Table 1 we summarized some of these gene mutations that are described below:

Table 1

The gene polymorphisms studied according to NCBI (www.ncbi.nlm.nih.gov).

Coded protein	Gene	Chromosome location	Studied SNPs	Change at DNA level	Change at Amino acid level
Blood coagulation Factor V	FV	Chromosome 1 (1q24.2)	rs 6025 (FVLeiden)	G1691A (R506Q)	Arg506Gln
			rs1800595 (FV HR2)	A4070G	His1299Arg
Blood coagulation Factor II (Prothrombin)	FII	Chromosome 11 (11p11.2)	rs1799963	G20210A	N/A
5, 10 Methylene tetrahydrofolate Reductase	MTHFR	Chromosome 1 (1p36.22)	rs1801133	C677T	Ala 222 Val
			rs1801131	A1298C	Glu429Ala
Blood coagulation Factor XIII	F XIII A1	Chromosome 6 (6p25.1)	rs 5985	G103T	Val 34 Leu
Plasminogen Activator Inhibitor 1 (PAI-1)	Serpine1	Chromosome 7 (7q22.1)	rs1799762	4G/5G1-bp guanine deletion/insertion (4G/5G) in the promoter region	N/A
Endothelial Protein C Receptor (EPCR):	EPCR (PROCR)	Chromosome 20 (20q11.22)
Haplotype 1 (A1)			rs 9574	G4678C	N/A
Haplotype 3 (A3)			rs 867186	A4600G	Ser219Gly

N/A: No available data.

FV Leiden (G1691A) and FV HR2 (H1299R) gene polymorphisms have been previously reported as risk factors closely related to hereditary thrombophilia. Individuals carrying FV LeidenG1691A mutations are at high risk of thrombosis through its enhancement of activated protein C resistance [12].

ProG20210A mutation (Prothrombin) is the second most common cause of hereditary thrombophilia after the Factor V Leiden mutation through its effect on plasma levels of prothrombin [13].

It has been established that individuals who have a Plasminogen Activator Inhibitor 1 (PAI-1) gene mutation have high levels of plasma PAI-1 that may cause impaired fibrinolysis. The 4G allele of PAI-1 gene polymorphism is considered to be a high risk factor for thromboembolic events such as deep vein thrombosis [14] and myocardial infarction [15],while it has been suggested that the protective role of the FXIII V34L variant against VTE is attributable to a high genotype/allele frequency of FXIII V34L polymorphism in healthy individuals who have altered FXIII activity and fibrin clot stability [16].

MTHFR C677T and MTHFR A1298C variants of the methylene tetrahydrofolate reductase (MTHFR) gene have been widely studied as one of the risk factors for hereditary thrombophilia. MTHFR is an essential enzyme responsible for conversion of 5,10-methylenetetrahydrofolate to 5-methyl tetrahydrofolate and homocysteine production. It was found that carriers of the T allele of MTHFR C677T, whether coupled with the C allele of the MTHFR A1298C gene polymorphism or not, are associated with reduced MTHFR enzyme activity in combination with elevated plasma homocysteine levels. These changes may precipitate an impaired vascular tone and coagulability state [17]. However, the association between MTHFR mutation and hereditary thrombophilia is controversial.

The EPCR (Endothelial Protein C Receptor) gene, also known as PROCR (protein C receptor gene), encodes a protein receptor (EPCR) that is crucial for the protein C activation involved in the blood coagulation cascade. Among its four identified haplotypes, the A3 haplotype is found to be associated with venous thrombosis [18].

Undeniably, the primary cause of an increased tendency to vascular thrombosis in KS patients is not clearly understood, but hereditary thrombophilia is considered one of the precipitating factors for VTE in men with KS. The aim of this study was to investigate the genotype and allele frequencies of common gene polymorphisms related to hereditary thrombophilia in infertile males with KS compared to healthy males.

Materials and methods

Study subjects

The present study included 85 newly diagnosed, untreated, infertile males with KS, referred from the Andrology Outpatient Clinic, Faculty of Medicine, Alexandria University, Egypt, between February 2017 and April 2018. In addition, 75 age and ethnicity matched healthy males who had at least one normal child were also selected as a control group. History, clinical examination and laboratory investigations were done.

Laboratory investigations included fasting blood sugar and serum lipid profile (LDL, HDL, Cholesterol and triglycerides) on Beckman Coulter analyzers, USA. Roche Elecsys assays were used to assess serum hormone levels of FSH, LH, Total Testosterone (TT) and Estradiol by the electrochemiluminecsence method on a Cobase 411 analyzer (Roche Diagnostics GmbH, Germany) with limits of detection (LOD) of <0.100 mIU/mL, 0.100 mIU/mL, 0.025 ng/mL and 18.4 pmol/L respectively. Established reference ranges were as follows: FSH: 1.3-12.4 mIU/mL, LH:1.7–8.6 mIU/mL, TT: 2.49–8.36 pg/ml and Estradiol: 27.9–156.38 pmol/L.

Chromosome analysis from peripheral blood using trypsin and giemsa stain, according to Seabright with modifications (1971), was carried out on all participants to confirm the diagnosis and exclude other chromosome aberrations [19].

KS patients with a chromosomal abnormality other than 46,XXY or chromosomal mosaicism were excluded. For the control group, individuals with a history of thromboembolic events or hormonal disturbances were excluded and only men with a normal male karyotype (46,XY) were included in the study. The number of healthy men excluded from the control group based on presence of VTE events was not available.

Informed written consent was obtained from all participants. Approval of the study by the ethical committee of the Faculty of Medicine, University of Alexandria, Egypt was acquired. The study protocol was consistent with the guidelines of the Declaration of Helsinki, 1964.

Molecular genetic analysis

DNA extraction

3 ml blood samples were collected from all participants for DNA extraction using Quick-DNA Miniprep Kit (ZymoResearch, USA, Cat.No.3024) according to the manufacturer’s instructions. The purity and concentration of extracted DNA were measured by a NanoDrop TM 1000 spectrophotometer and stored at −20 °C for future use.

Genotyping

Genotyping was run for nine well-known thrombophilic gene polymorphisms using the Multiplex PCR reverse-hybridization method in combination with CVD Strip Assays, Vienna Lab Diagnostics GmbH ®(REF. 4-360).

The gene polymorphisms studied were summarized in Table 1 according to data provided by the National Center for Biotechnology Information (NCBI).

DNA amplification was carried out on an Applied Biosystem Thermal Cycler 2720. PCR conditions were as follows: A Pre-PCR step was run at 94 °C for2 min. followed by 35 cycles of 94 °C for 15 sec. then 58 °C for 30 sec., followed by 72 °C for 30 sec., then a final extension at 72 °C for 3 min. Finally, the amplification products were selectively hybridized to allele-specific oligonucleotide probes immobilized on a test strip. Mutations were detected by enzymatic color reaction visible to the naked eye and the interpretation of genotypes was determined according to the enclosed Collector TM sheet.

One of three genotypes for each polymorphism was obtained for every individual: normal, heterozygous, or homozygous. EPCR alleles were A1 (H1), A2 (H2), A3 (H3), and so the resulting genotypes could be: A1/A1, A1/A2, A1/A3, A2/A2, A2/A3, A3/A3.

Statistical analysis

Data were computerized and analyzed using an IBM SPSS software package version 20.0 (Armonk, NY:IBM Corp). Student t-test was used to compare the two groups for normally distributed quantitative variables. Chi-square and Fisher’s exact test were used for qualitative variables. Genotype and allele frequency were calculated by direct counting. Comparisons between groups were done using Chi-square test, Fisher’s exact test, or Monte Carlo, according to data obtained. The risk of association between different genotypes and hereditary thrombophilia in KS and control men was assessed by odds ratio and 95% Confidence Interval (OR (95% CI)). The significance of the results obtained was judged at the 5% level.

Results

Table 2 illustrates the clinical and laboratory characteristics of the KS patients in comparison to the control group. A highly significant difference was found between both groups regarding fasting blood sugar (P < 0.001), lipid profile (P < 0.001) and hormonal assay (P < 0.001).

Table 2

Clinical and laboratory characteristics of KS patients and control.

	KS (n = 85)	Control (n = 75)	P value
Age (years)	30.75 ± 6.93	30.25 ± 8.96	0.6901
BMI (kg/m2)	27.23 ± 4.12	22.8 ± 2.61	<0.001*
Diabetes mellitus	5(5.9%)	0(0.0)	0.061
Hypertension	3(3.5%)	0(0.0)	0.248
History of previous thrombosis	0(0.0)	0(0.0)	–
History of varicose veins	6(7.1%)	2(2.7%)	0.284
Fasting blood sugar (mg/dl)	88.7 ± 4.5	85.6 ± 2.5	<0.001*
Systolic blood pressure (mmHg)	135 ± 22	96 ± 20	<0.001*
Diastolic blood pressure (mmHg)	88 ± 8.71	87.5 ± 11	0.749
Serum cholesterol (mg/dl)	190.6 ± 17.2	156.5 ± 13.36	<0.001*
Serum triglycerides (mg/dl)	139.2 ± 16.1	87.6 ± 14	<0.001*
LDL (mmol/l)	117.6 ± 18.2	92.1 ± 13.2	<0.001*
HDL (mmol/l)	15.8 ± 7.6	24.5 ± 9.3	<0.001*
Testosterone (ng/ml)	1.08 ± 0.25	6.04 ± 2.30	<0.001*
FSH (mIU/ml)	23.7 ± 2.5	3.74 ± 0.21	<0.001*
LH (mIU/ml)	20.3 ± 1.5	3.39 ± 0.25	<0.001*
Estradiol (pmol/L)	116.4 ± 8.52	122.1 ± 6.45	<0.0001*

BMI: Body mass index, LDL: Low density lipoprotein, HDL: High density lipoprotein, FSH: Follicle stimulating hormone, LH: Luteinizing hormone.

*Statistically significant at p < 0.05.

None of the KS patients had a thromboembolic event at the time of the study. Chromosome analysis of clinically suspected KS cases was 47, XXY karyotype.

Table 3 and Table 4 illustrate the genotype and allele frequency in both KS cases and controls.

Table 3

Genotype frequency of the thrombophilia gene polymorphisms in patients with KS compared to control group.

Genotype		Cases (n = 85)	Control (n = 75)	OR (95%C.I)	P value
FV G1691A (Leiden)
GG*		45(52.9%)	64(85.3%)	Reference	<0.001
GA		40(47.1%)	11(14.7%)	5.172(2.398–11.154)
AA		0(0%)	0(0%)	–
FV H1299R
AA*		3(3.5%)	45(60%)	Reference	<0.001
AG		82(96.5%)	30(40%)	41.0(11.849–141.86)
GG		0(0%)	0(0%)	–
Pro G20210A
GG*		71(83.5%)	67(89.3%)	Reference	0.359
AG		14(16.5%)	8(10.7%)	1.651 (0.651–4.188)
AA		0(0%)	0(0%)	–
MTHFR C677T
CC*		49(57.6%)	51(68%)	Reference	0.178
CT		36(42.4%)	24(32%)	1.561(0.816–2.986)
TT		0(0%)	0(0%)	–
MTHFR A1298C
AA*		42(49.4%)	33(44%)	Reference	0.494
AC		43(50.6%)	42(56%)	0.804(0.431–1.500)
CC		0(0%)	0(0%)	–
Factor XIII V34L
GG*		51(60%)	32(42.7%)	Reference	0.035
GT		34(40%)	42(56%)	0.507(0.269–0.955)
TT		0(0%)	1(1.3%)	–
PAI-1 4G/5G
5G/5G*		15(17.6%)	32(42.7%)	Reference	0.013
4G/5G		46(54.1%)	38(50.7%)	2.583(1.221–5.461)
4G/4G		24(28.2%)	5(6.7%)	10.240(3.267–32.087)
EPCR Haplotype
A1/A1		23(27.1%)	22(29.3%)	Reference	–
A1/A2		26(30.6%)	22(29.3%)	1.130(0.500–2.554)	0.768
A1/A3		0(0%)	0(0%)	–	–
A2/A2		24(28.2%)	23(30.7%)	0.998(0.441–2.261)	0.996
A2/A3		6(7.1%)	8(10.7%)	0.717(0.214–2.404)	0.590
A3/A3		6(7.1%)	0(0%)	–	–

*Normal homozygous genotype, OR: Odds ratio, CI: Confidence Interval, statistically significant at p < 0.05.

Table 4

Allele frequency of the thrombophilia gene polymorphisms in patients with KS compared to control group.

	Cases (n = 85)	Control (n = 75)	OR (95%C.I)	P value
FV G1691A (Leiden)
G	130(0.765)	139(0.927)	3.888(1.913–7.899)	<0.001
A*	40(0.23)	11(0.07)
FV H1299R
A	88(0.518)	120(0.80)	3.727(2.259–6.148)	<0.001
G*	82(0.48)	30(0.20)
Pro G20210A
G	156(0.918)	142(0.947)	1.593(0.649–3.909)	0.309
A*	14(0.082)	8(0.053)
MTHFR C677T
C	134(0.788)	126(0.84)	1.410(0.796–2.496)	0.238
T*	36(0.212)	24(0.16)
MTHFR A1298C
A	127(0.74.7)	108(0.72)	0.871(0.529–1.431)	0.585
C*	43(0.253)	42(0.28)
Factor XIII V34L
G	136(0.80)	106(0.707)	0.602(0.36–1.007)	0.053
T*	34(0.20)	44(0.293)
PAI-1 4G/5G
5G	76(0.447)	102(0.68)	2.628(1.664–4.152)	<0.001
4G*	94(0.553)	48(0.32)
EPCR Haplotype
A1	72(0.424)	66(44)	Reference	0.114
A2	80(0.471)	76(0.507)	0.965(0.610–1.526)
A3*	18(0.106)	8(0.053)	2.063(0.841–5.059)

* Mutant allele (MAF: Minor allele frequency), OR: Odds ratio, CI: Confidence Interval, statistically significant at p < 0.05.

FV Leiden and FV HR2 Polymorphisms

Significant differences in heterozygous and normal homozygote genotypes between KS cases and control were noticed for both polymorphisms (P < 0.001), while mutant homozygous genotypes (AA for FV Leiden and GG for FV HR2 gene polymorphisms) were not found in KS males or controls. Mutant alleles of both polymorphisms were significantly higher in KS men than the control (P < 0.001).

Prothrombin G20210A Polymorphism

The frequency of GG and AG genotypes in KS patients was 83.5% and 16.5% versus 89.3% and 10.7% in the control, while the AA genotype was not found in either group (P = 0.359). The allele frequency was not significantly elevated in KS men (P = 0.309).

PAI-1 4G/5G gene polymorphism

The frequency of 4G/5G genotype was 54.1% in KS men and 50.7% in control men. This increase in the KS patients was statistically significant (P = 0.013). We also found a statistically significant difference in the 4G/4G genotype frequency in KS patients compared to the control group (28.2% versus 6.7%, P < 0.001). The frequency of mutant 4G alleles was significantly higher in KS men than the control (0.55 versus 0.32, P < 0.001).

Factor XIII V34LPolymorphism

Despite a significant difference in the frequencies of GG, GT, and TT genotypes of Factor XIII V34L polymorphism between KS men and control men (P = 0.035), the difference in the mutant allele (T) frequency between both groups was statistically insignificant (P = 0.053).

MTHFR gene polymorphisms

There was no statistically significant difference in genotype and allele frequency of MTHFR C677T and MTHFR A1298C polymorphisms in either group. Mutant homozygotes were absent in both groups.

EPCR polymorphism

No significant difference was found between the groups regarding the genotype frequency of EPCR polymorphism. We found the A3/A3 genotype in 7.1% of KS men but absent in the control. Again, the frequency of the A3 allele was more common in KS men than in the control (0.106 versus 0.053) with odds ratio of 2.063 (95%CI 0.841 to 5.059), but this difference was statistically insignificant. (P = 0.114).

Combined mutant alleles

In Table 5, we analyzed the data obtained concerning the frequency of male carriers of coexisting mutant alleles of the different polymorphisms studied. It was found that 90.7% of control men had <3 mutant alleles in comparison to 43.5%of KS men, while 50.6% of KS men were carriers of 3–5 mutant alleles as opposed to9.3% of the control men (P < 0.001). Five KS patients (5.9%) had more than 5 mutant alleles in comparison to none in the control group (P < 0.001).

Table 5

Frequency of combined mutant alleles in KS patients and control.

Number of alleles	Cases (n = 85)		Control (n = 75)		P value	OR (95%C.I)
	No.	%	No.	%
<3	37	43.5	68	90.7	<0.001	0.079(0.033–0.192)
3–5	43	50.6	7	9.3	<0.001	9.946(4.098–24.137)
>5	5	5.9	0	0	–	–

OR: Odds ratio, CI: Confidence Interval, statistically significant at p < 0.05.

Discussion

The tendency towards thromboembolism is a serious complication related to KS. Some KS patients may suffer from VTE in the form of deep vein thrombosis or pulmonary embolism, or both [20] while others may complain of recurrent venous ulcers [21], [22]. The primary cause of this increased risk of VTE in KS patients is not clearly understood [8] and so this has encouraged scientists to look for the cause and whether it is an outcome of an underlying genetic susceptibility, hormonal disturbances, or both.

In this study, screening for hereditary thrombophilia was done using a broad panel of six genes known to be established risk factors for thromboembolism and which, to the best of our knowledge, have never been assessed before in infertile men with KS.

Based on studies published previously, the relative risk for venous thrombosis in carriers of FV Leiden mutation is estimated to be three to eight fold [12] and two to five fold in carriers of Pro G20210A mutation. In carriers of both Pro G20210A and factor V Leiden mutations, the risk may even rise to nine fold [13], [23]. In the current study, a high percentage of KS men were carriers of FV Leiden (G1691A), FV HR2 (H1299R) and Pro G20210A gene mutations and this should alert us to the possibility of the occurrence of such thrombotic events. Unfortunately, we have no data on the follow up for such cases and a large prospective study is needed to confirm these findings.

In case of PAI-14G/5G gene polymorphism, KS carriers of the 4G/5G variant may have increased plasma levels of PAI-1 which, in the presence of an underlying hypercoagulable state associated with Klinefelter syndrome, could increase the risk of vascular thrombosis [24], [25]. High plasma levels of PAI-1 may impair the fibrinolysis cascade provoked by the underlying hormonal imbalance and result in an increased tendency to vascular thromboembolism [15], [26].

Regarding the FXIII V34L polymorphism, the minor allele frequency (T allele) was 0.29 in controls recruited from Egypt, which falls within the range (0.01–0.4) reported in different ethnic groups [27]. Here, a protective role for the T allele of FXIII V34L polymorphism against vascular thrombosis could be suggested (OR was 0.602), but even so, a detailed population study is needed to confirm this.

In addition, it has been reported that the H3 (A3) haplotype of the EPCR gene is a potential risk factor for venous thrombosis through its association with high levels of a soluble form of EPCR that results in inhibition of APC [28], [29] while a protective role for the A1 haplotype has been suggested [30], [31]. Indeed, in the present study we found a high frequency of the H3 haplotype in KS males compared to controls and theA3/A3 genotype was absent in healthy men. By literature review, it is the first time to assess the prevalence of EPCR haplotypes in infertile KS males. However, this difference was insignificant which could be attributed to gene-gene interactions, sample size, or both.

Furthermore, the coincidence of more than one mutant allele of different thrombophilic gene polymorphisms was more evident in infertile men with KS than the controls. In view of published reports regarding this issue, we assume that KS patients who carry the combined heterozygous genotypes of such genes are at high risk of developing thromboembolism, and this is probably caused by an interaction between genetic products that share the same biological pathway and which may result in impaired fibrinolysis or coagulation cascades, or both [32], [33], [34].

Biases that may result from the underlying genetic background were minimized in the present study by sample size in conjunction with matched ethnicity between patients and control, which strengthened the current work. The main limitation was the absence of VTE events in both groups, which rendered the sample of control men not representative of the general population and so the susceptibility of KS men to VTE could not be assessed.

Conclusion

The results reached during this study make clear that heterozygote genotypes, compound genotypes, and mutant alleles of several key thrombophilic genes are found in high frequency along the genetic profile of infertile KS males. Further research is recommended on large cohorts in order to investigate the genotype-phenotype correlation in KS syndrome in an attempt to provide proper management and genetic counseling to high risk families.

Declarations

Ethics approval and consent to participate

The study was approved by the ethics committee of the Faculty of Medicine, Alexandria University (IRB NO. 00007555- FWA NO.00018699). An informed written consent was obtained from all participants in the study according to the Declaration of Helsinki 1964 and its extended demands.

Consent for publication

Consent for publication was obtained from all authors to the Journal of Clinical and Translational Endocrinology.

Availability of data and material

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.