Increased risk of the abdominal aortic aneurysm in carriers of the MTHFR 677T allele.

Abstract

Abdominal aortic aneurysm (AAA) presents itself as a progressive dilation of the abdominal aorta, leading--if untreated--to rupture. It is a common disease of the elderly, with a complex etiology. Several genetic, biochemical and environmental factors are recognized as relevant for the pathogenesis of AAA. We determined the polymorphism of the MTHFR (methylenetetrahydrofolate reductase) gene within the fourth exon (C677T) in 63 patients with AAA and compared it to that in 75 subjects of the population sample. The frequencies of the C/C, C/T and T/T genotypes were 65%, 27%, and 8% in the population sample and 33%, 60%, and 6% in the patients. This corresponds to a 4.4-fold greater risk of AAA in subjects who have the 677C/T variant of MTHFR, as compared with those who are 677C/C (p < 0.0001; 95% CI=2.11-9.34). The frequency of allele MTHFR 677T in patients (0.37) was higher than in the population sample (0.21; p < 0.007). This association between the common allele of the MTHFR gene--MTHFR 677T--and the development of AAA suggests that elevated homocysteine (Hcy) may disturb the function of the aortic wall. The disturbance may involve enhancement of elastin degradation, the process enhanced by mild hyperhomocysteinemia in minipigs. The magnitude of this effect, which refers to the AAA patients unselected for familial occurrence, indicates that the disturbance of aortic wall physiology caused by the presence of the MTHFR 677T allele is greater than the effect of the earlier described allele disequilibrium at the polymorphic alleles of the PAI1 (plasminogen activator inhibitor 1) gene seen only in familial cases of AAA.

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J. Appl. Genet. 44(1), 2003, pp. 85-93
Increased risk of the abdominal aortic aneurysm
in carriers of the MTHFR 677T allele
Ewa STRAUSS1, Krzysztof WALISZEWSKI2, Marcin GABRIEL2, Stanis³aw ZAPALSKI2,
Andrzej L. PAWLAK1
1Institute of Human Genetics, Polish Academy of Sciences, Poznañ, Poland
2Department of General and Vascular Surgery, Institute of Surgery, Karol Marcinkowski
University of Medical Sciences, Poznañ, Poland
Abstract. Abdominal aortic aneurysm (AAA) presents itself as a progressive dilation
of the abdominal aorta, leading – if untreated – to rupture. It is a common disease of
the elderly, with a complex etiology. Several genetic, biochemical and environmental
factors are recognized as relevant for the pathogenesis of AAA. We determined
the polymorphism of the MTHFR (methylenetetrahydrofolate reductase) gene within
the fourth exon (C677T) in 63 patients with AAA and compared it to that in 75 subjects
of the population sample. The frequencies of the C/C, C/T and T/T genotypes were
65%, 27%, and 8% in the population sample and 33%, 60%, and 6% in the patients.
This corresponds to a 4.4-fold greater risk of AAA in subjects who have the 677C/T
variant of MTHFR, as compared with those who are 677C/C (p<0.0001; 95%
CI=2.11-9.34).ThefrequencyofalleleMTHFR677Tinpatients(0.37)washigherthan
inthepopulationsample(0.21;p<0.007).Thisassociationbetweenthecommonallele
of the MTHFR gene – MTHFR 677T – and the development of AAA suggests that ele-
vated homocysteine (Hcy) may disturb the function of the aortic wall. The disturbance
may involve enhancement of elastin degradation, the process enhanced by mild
hyperhomocysteinemia in minipigs. The magnitude of this effect, which refers to
the AAA patients unselected for familial occurrence, indicates that the disturbance of
aorticwallphysiologycausedbythepresenceoftheMTHFR677Talleleisgreaterthan
the effect of the earlier described allele disequilibrium at the polymorphic alleles of
thePAI1(plasminogenactivatorinhibitor1)geneseenonlyinfamilialcasesofAAA.
Key words: abdominal aortic aneurysm, elastin, homocysteinemia, MTHFR gene, MTHFR
677T allele, polymorphism.
Received: July 18, 2002. Accepted: October 22, 2002.
Correspondence: E. STRAUSS, Institute of Human Genetics, Polish Academy of Sciences,
ul. Strzeszyñska 32, 60-479 Poznañ, Poland, e-mail: strauss@icpnet.pl

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Introduction
Abdominal aortic aneurysm (AAA) is characterized by a progressive dilation of
the abdominal aorta and thinning of the vessel wall secondary to the degradation
ofthemedialayerandthefragmentationofelasticandcollagenousfibersdecreas-
ingtheirdensityinthistissue(RAMSBOTTOMetal.1994).Ultrasonographyiscur-
rently the most precise and commonly available method of early detection
of AAA. Its prevalence in adult autopsy series varies between 1% and 6%
(JOHNSON et al. 1985). In a recent international study of ASHTON and CASS
group (2001) – the highest age-standardized AAA prevalence was recorded
in the UK (7.7%) and the lowest in Denmark (4.5%). About 20% of AAA cases
occur in families in which two or more persons are affected. The increased fre-
quency of the PAI1 gene allele 5D was noted in familial cases of AAA, but not in
the remaining patients. This was considered as an indication that the pathogenesis
may be distinct in the familial versus the sporadic cases of AAA (ROSSAAK et al.
2000). The female patients of AAA occur more frequently in the familial type of
thedisease(VERLOESetal.1995).ThepatientswithAAAhaveplasmaHcylevels
significantly higher than the normal values (BRUNELLI at al. 2000).
The current study was established to determine the frequency of the MTHFR
(methylenetetrahydrofolate reductase) 677T allele in 63 patients (5 women
and 58 men) with AAA, as compared to that in 75 subjects of the population sam-
ple (26 women and 49 men).
Material and methods
Subjects studied
The group with AAA consisted of 63 patients (5 women and 58 men; mean age
64.9 ±7.9 years; age range 46-81). The diagnosis was confirmed by
ultrasonography. The mean diameter of abdominal aorta was 6.0 ±1.6 cm (size
range 3-10 cm). In all patients the abdominal aortic aneurysmectomy was per-
formed. The population sample consisted of 75 subjects (26 women and 49 men)
randomly selected from a local population. Patients and the population group
were selected from the inhabitants of downtown Poznañ. In the control group
males and females did not differ when the MTHFR allele frequencies were com-
pared (data not shown). Classic markers of the risk factors of vascular disease
were determined in 51 of 63 AAA patients. The occurrence of other diseases,
smoking habits and occurrence of overweight in the group of AAA patients were
recorded. Overweight was defined as BMI (Body Mass Index, BMI [kg/m2]
= body weight [kg]/(height [m])2) above 30.
Genotyping of MTHFR. Genomic DNA was isolated from peripheral blood
leukocytes by the phenol extraction method. MTHFR polymorphism within
86E. Strauss et al.

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thefourthexon(C677T)wasascertainedbythePCR-RFLP(polymerasechainre-
action, restriction fragment length polymorphism) method (FROSST at al. 1995).
This single nucleotide substitution results in a conversion of alanine to valine in
the MTHFR (protein product). The amplified PCR fragment was digested with
the HinfI restriction enzyme, which can recognize C to T substitution.
If the MTHFR 677T allele is present, HinfI digests the 198-bp fragment into
a 175-bp and 23-bp fragment. The fragments were resolved by electrophoresis in
10%polyacrylamidegelsandstainedwithethidiumbromide.Genotypeswereex-
pressed as C/C for homozygous normal, C/T for heterozygous, and T/T for homo-
zygous variant.
Other laboratory studies
Fasting serum triglyceride (TG), total serum cholesterol (TC), LDL-cholesterol
(low density lipoprotein; LDL-C) and HDL-cholesterol (high density lipoprotein,
HDL-C)weredeterminedbyroutinelaboratoryassays.Whendirectmeasurement
was not available, LDL-C levels were calculated from TC, TG and HDL-C by us-
ing Friedewald’s formula (LDL-C [mmol/l] = TC [mmol/l] – HDL-C [mmol/l] –
0.45 × TG [mmol/l]; TG<4.6 mmol/l) (CHOTKOWSKa et al. 2001, KRAUSE et al.
1996).
Statistical analysis
Results of continuous data (e.g. cholesterol) are presented as mean values ±SD
(standard deviations). Frequencies of MTHFR alleles and genotypes were com-
pared between the study and the population groups by using a two-tailed Fisher’s
exact test. Odds ratio (OR) and 95% confidence interval (CI) were calculated on
the basis of logistic regression analysis.
Results
Characteristics of the study population
Table 1 present the serum lipid parameters in AAA patients, compared to
the range of normal values for the Polish population (CYBULSKA et al. 1994).
The studied group displays increased median values of total cholesterol. Most
(34)ofthepatientssmoke(67%).In9(18%)ofAAApatients,increasedvaluesof
Body Mass Index (BMI>30) were noted. Heart diseases were diagnosed in 24
(47%) and diabetes in 4 (7%) of the AAA patients.
Frequency of the MTHFR 677T allele
GenotypingresultsaregiveninTable2.Inthegroupof63patientsdiagnosedwith
AAA,theMTHFR677Tallelefrequencywas0.37(Table2A),whereasin75sub-
MTHFR 677T allele in abdominal aortic aneurysm 87

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Table 1. Serum lipid parameters (mean values and SD) in AAA patients
Serum lipid parameter
AAA patients
(51)
Range of normal values
Total cholesterol (TC) (mmol/l)
5.8 ±1.1
<5.2
HDL cholesterol (mmol/l)
0.96 ±0.23
>1.0(F); >0.8(M)
LDL cholesterol (mmol/l)
3.74 ±0.88
<4.0
Triglycerides (mmol/l)
1.8 ±0.8
<2.0
Table 2. A) The numbers and frequencies of MTHFR alleles in AAA patients and
thepopulationsample.B)MTHFRgenotypesinAAApatientsandthepopulationsample.
C) MTHFR genotypes in male AAA patients and males of the population sample.
Adjustedoddsratios(ORs)and95%confidenceintervals(CIs)forgenotypes677C/Tand
677 C/T + 677 T/T, using 677 C/C as a reference.
A)
MTHFR allele
Number of MTHFR alleles
population sample
N = 75 (%)
AAA patients
N = 63 (%)
677 C
118 (79) 80 (63)
677 T
32 (21)a
46 (37)a
ap<0.007 (by Fisher’s exact test)
B)
MTHFR genotype
Number of persons
OR95% CI
population sample
N = 75 (%)
AAA patients
N = 63 (%)
677 C/C
49 (65)
20 (27)a
21 (33)
38 (60)a
1–
677 C/T
4.432.11-9.34
677 T/T
6 (8)
26 (35)b
4 (6)
42 (67)b
ns ns
677 C/T+T/T
3.771.86-7.65
ap<0.0001,bp<0.0003 (by Fisher’s exact test)
C)
MTHFR genotype
Number of males
OR95% CI
population sample
N = 49 (%)
AAA patients
N = 58 (%)
677 C/C
32 (65)
13 (27)a
20 (34)
35 (60)a
1 -–
677 C/T
4.311.85-10.05
677 T/T
4 (8)
17 (35)b
3 (5)
38 (66)b
nsns
677 C/T+T/T
3.581.61-7.96
ap<0.001,bp<0.002 (by Fisher’s exact test)

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jects of the population sample this value was significantly lower, amounting to
0.21 (p<0.007). The frequencies of the C/C, C/T and T/T genotypes among
the subjects of the population sample were: 65%, 27%, and 8%. The correspond-
ing frequencies among the patients with AAA were: 33%, 60%, and 6%. We ob-
servedasignificant4.4-foldincreaseintheriskofAAAamongsubjectswhohave
the 677 C/T variant of MTHFR, as compared with those who are 677 C/C
(p < 0.0001; 95% CI = 2.11-9.34). The presence of the C677T substitution in one
or both alleles was associated with a 3.8-fold increase in the risk of AAA
(p<0.0003;95%CI=1.86-7.65;Table2B).FollowingtheanalysisofMTHFRal-
lelefrequenciesinthemalepatientscomparedtothemalemembersofthepopula-
tionsample,therespectiveORvalueswere4.31(for677C/TvsC/C)and3.58(for
677 C/T+T/T vs C/C) (Table 2C).
Discussion
In this study we report the association between the susceptibility to AAA
and the allele 677T of the MTHFR gene. The data presented in Table 2A indicate
that the risk of AAA is strongly associated with the MTHFR 677T allele. In the
group of AAA patients (unselected for familial occurrence) an increase in the fre-
quencyofMTHFR677Tallelewasnoted,ascomparedtothepersonsofthepopu-
lation sample (p < 0.007, Table 2A). By contrast, the increase in the frequency
of the 5G allele of the plasminogen activator inhibitor 1 gene (PAI1 5G), reported
byROSSAAKetal.(2000),wasnotedonlyinfamiliarcasesofAAAandatalower
magnitude(p<0.03).Sincethepersonshomozygousforthe5GalleleofthePAI1
gene display the higher aneurysm growth rate, whereas the postrepair mortality in
thesecasesislower,therelationsbetweenthePAI15Galleleandthepathogenesis
of AAA may be considered rather complex (JONES et al. 2002). The effect
of the MTHFR 677T allele on the risk of AAA is probably more straightforward,
as compared to that of PAI1 5G.
The frequency of the heterozygous genotype was greater among the patients
withAAAthaninsubjectsofthepopulationsample,andthedifferencewashighly
significant (p < 0.0001; OR = 4.4; 95% CI = 2.11-9.34; Table 2B). The MTHFR
677T allele and the 677 C/T genotype frequencies in the studied population group
were highly concordant with the corresponding values obtained in the larger
(310 persons) population group from the Cracow district of Poland (SZCZEKLIK
et al. 2001). An unexpected finding was the absence of the increase in the fre-
quency of MTHFR 677 T/T persons in the AAA group. This may be related to al-
leledisequilibriumbetweenthisalleleandtheMTHFR1298Callele,describedby
VANDERPUTetal.(1998).Theobservationspresentedindicatethatthiscommon
MTHFR variant, which produces a mild elevation of plasma homocysteine, pre-
disposes to AAA.
MTHFR 677T allele in abdominal aortic aneurysm 89

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The etiology of aortic aneurysm formation is considered to be distinct from
thatofatherosclerosis(ROSSAAKetal.2000).InpatientswithAAA,significantly
elevated Hcy plasma levels were found (BRUNELLI et al. 2000). Hcy is
a non-protein amino acid, which is involved in the one-carbon metabolism and
methylation reactions. The high plasma levels of Hcy are toxic to the vascular
cellsandtissues,andthenareanimportantriskfactorinvasculardisease.Thetwo
major ways of Hcy detoxification are: the trans-sulphuration pathway and
the re-methylation pathway (ROSENBLATT 1989). CHEN et al. (1999) reported
that the trans-sulphuration pathway is absent from the human cardiovascular sys-
tem, which may sensitize these tissues to Hcy toxicity. Then in this tissue the me-
tabolism of Hcy largely depends on remethylation reaction, which is catalysed by
methionine synthase (MS). In this reaction the 5-methyltetrahydrofolate
(5-MetTHF)servesasamethyldonor.Thiscompoundisproducedintheirrevers-
ible reaction catalysed by MTHFR (EC 1.5.1.0). Consequently, the genetic defi-
ciency of this enzyme results in excessive accumulation of Hcy, which
particularlyaffectsthevascularsystem.TheMTHFR677Talleleproducestheen-
zymewithreducedactivityandincreasedthermolability(KANGetal.1991)andis
implicitly associated with mild hyperhomocysteinemia.
The MTHFR C677T polymorphism is common in all studied ethnic groups;
however its frequency varies in a wide range. The highest frequencies
of the low-activity thermolabile allele MTHFR 677T up to 0.38 was reported in
FrenchCanadians(FROSSTetal.1995)whereasthelowest(0.1)inAfricanAmer-
icans (MCANDREW et al. 1996). The above-mentioned ethnic high frequency
of the thermolabile variant of the MTHFR gene for Caucasians (0.25-0.38) coin-
cides with the high frequency of AAA in Caucasian males (4.5%-7.7%). This
compares to the low frequency of MTHFR 677T (0.1) in African American males
and the parallel low frequency of AAA in this ethnic group (1.5%) (JOHNSON
et al. 1985, LAMORTE et al. 1995).
To investigate in vivo the effects of hyperhomocysteinemia on the aortic wall,
CHEN et al. (2001) generated mthfr knockout mice. Plasma total homocysteine
levels in mthfr +/– and mthfr –/– mice were respectively 1.6- and 10-fold higher
than those in wild-type littermates. At older age, abnormal lipid deposition in
theproximalportionoftheaortawasobservedbothinheterozygotesandinhomo-
zygotes, pointing to the involvement of hyperhomocysteinemia in pathologic
changes in large vessels.
In vitro tissue culture studies show that Hcy tiolactone induces
thecaspase-independentvascularendothelialcelldeathwithfeaturesofapoptosis
(MERCIE et al. 2000). In Saccharomyces Hcy is converted by methionyl-,
isoleucyl-, and leucyl-tRNA synthetases to the thioester Hcy thiolactone.
Thehighenergycostsofthisprocessaredetrimentaltocellgrowth(JAKUBOWSKI
1991). Under physiological conditions Hcy thiolactone easily reacts with
side-chain amino groups of lysine residues in proteins (JAKUBOWSKI 1999).
90E. Strauss et al.

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The pathogenicity of Hcy through formation of Hcy thiolactone or stimulation
ofproductionofoxygenradicalscanbepreventedbyitsS-nitrosylationwithnitric
oxide to S-nitroso-Hcy. Methionyl-tRNA synthetase binds S-nitroso-Hcy to
tRNAMet, which leads to translational incorporation of S-nitroso-Hcy into protein
(JAKUBOWSKI 2001). However, the NO-producing endothelial cells are them-
selvessusceptibletothetoxicityofHcyandtherangeofthiscompensationislim-
ited. Translationally incorporated Hcy was also detected in the cultured human
vascular endothelial cells (STAMLER et al. 1993). Both translationally and
post-translationally incorporated Hcy was found in human blood proteins
(JAKUBOWSKI 2002).
The degradation of elastin, which is the major structural component of the aor-
tic wall, may be considered as the initial event in the formation of aneurysm
(WHITE et al. 1993). Elastin is a very stable protein with a biological half–life of
70 years. The basic regions of this protein are rich in lysine, which forms covalent
cross-links by extracellular oxidation (PATEL et al. 1995). Elastin is synthesized
and deposited in early childhood and no significant synthesis occurs in adult life.
Themainfactorthatcontributestothethinningoftheaorticwallwithage,appears
to be the intensity of the gradual loss of elastin from the aortic wall, which is not
compensated by the synthesis of this protein. Then, the age-related decrease in
elastin concentration is not due to the reduced transcription of the elastin gene.
Presumably, the degradation of elastin may be enhanced by translational incorpo-
ration of S-nitroso-Hcy and/or its N-homocysteinylation in the presence of Hcy
tiolactone.
The deterioration of the elastin structures of the media layer of the abdominal
aortawas found inminipigsfollowing
hyperhomocysteinemia (CHARPIOT et al. 1998). The presented observation of
the increased risk of AAA in carriers of the MTHFR 677T allele may then be due
to the enhancement of the processes of elastin degradation in these persons.
the dietary-inducedmild
Acknowledgments:ThisworkwassupportedbytheCommitteeforScientificRe-
search, Poland, grants: KBN 6.P05A.03921 (received by E.S.) and KBN 4.P05C.
01517 (received by K.W.).
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MTHFR 677T allele in abdominal aortic aneurysm 93