C677T polymorphism of the MTHFR gene
and variant hemoglobins: a study in newborn s
f rom Salvador, Bahia, Brazil
Polimorfismo C677T no gene da MTHFR
e hemoglobinas variantes: um estudo
em recém-nascidos de Salvador, Bahia, Brasil
1 Ce n t ro de Pe s q u i s a s Gonçalo Mo n i z , Fun dação Osw aldo Cruz, S a l va d o r, Bra s i l . 2 Faculdade de Fa r m á c i a , Un i versidade Fe d e ral da Ba h i a ,S a l va d o r, Bra s i l . 3 Faculdade de Me d i c i n a , Un i versidade Fe d e ral da Ba h i a ,S a l va d o r, Bra s i l .
C o r re s p o n d e n c e Marilda Souza Go n ç a l ve s La b o ratório de Patologia e Biologia Mo l e c u l a r, Ce n t ro de Pesquisas Gonçalo Mo n i z , Fundação Oswaldo Cruz. Rua Waldemar Falcão 121, S a l va d o r, BA
4 0 2 9 5 - 0 0 1 , Bra s i l . m a r i @ c p q g m . f i o c r u z . b r
Fábio David Couto 1
Elisângela Vitória Adorno 1 , 2 Joelma Fi g u e i redo Me n e zes 1 José Pe re i ra Mo u ra Neto 1
Ma rco Antônio Vasconcelos Rêgo 3 Mitermayer Galvão dos Reis 1 Marilda Souza Go n ç a l ves 1 , 2
A b s t r a c t
The C677T polymorphism in the methylenete-t ra h y d rofolamethylenete-te reducmethylenete-tase gene (MTHFR) is asso-ciated with an increase in total homocysteine serum levels (tHcy), described as a risk factor for c a rd i ovascular disease. Eight hundred fort y -t h ree neona-tes from -two differen-t ma-terni-ty h o s-p i t a l s , one s-public and another s-priva t e , in Sal-va d o r, Ba h i a , Brazil were screened for this poly-morphism by PCR and RFLP. The T-allele fre-quency in the total sample was 0.23, and the p re valence rates of heterozygous and homoz y-gous carriers were 36.2% and 5.3%, re s p e c t i ve l y. The T-allele frequency differed and the T/T g e n o-type was more pre valent at the private materni-ty hospital. The hemoglobin (Hb) profile was in-vestigated by HPLC in 763 new b o r n s . The fre-quency of variant Hb was higher at the public than at the private maternity hospital. The as-sociation of the C677T polymorphism and the Hb profile was investigated in 683 new b o r n s , s h owing a re l a t i vely high frequency of va r i a n t Hbs and the T allele. These data could prov i d e an important basis for further studies focusing on potential risks of va s o - o c c l u s i ve events in these individuals.
Newborns In f a n t ; Po l y m o r p h i s m ; He m o g l o b i-n o p a t h i e s
I n t ro d u c t i o n
Me t h y l e n e t e t ra h yd rofolate reductase (MTHFR) is a key enzyme in folate and homocysteine (Hcy) metabolism. A single point mutation c a u s-ing a C →T substitution at nucleotide 677 of the MTHFR gene has been associated with a t h e rmolabile enzyme form of low biological ac-tivity 1. High total homocysteine (tHcy) seru m
or plasma levels have been attributed to the p resence of the C677T MTHFR polymorphism, mainly in association with low folate and vita-min B12 levels 2 , 3 , 4. The role of high tHcy seru m
or plasma levels re c e i ved considerable intere s t after these factors we re implicated as an im-p o rtant risk factor for card i ovascular disease in a study examining childre n’s aorta fragments 5;
the study proposed that high homocysteine l e vels play a role in art e ri o s c l e rosis genesis.
Sickle-cell anemia (SS) is a monogenic dis-o rder with wdis-orldwide distributidis-on and high p re valence in Brazil 6. The mutant sickle
hemo-globin results from a single nucleotide substi-tution: (GAG →GTG) at the sixth codon of the beta-globin gene. Sickle cell anemia displays a h e t e rogeneous clinical presentation, unpre-dictable clinical development, and is chara ct e ri zed by chronic hemolyctic anemia and va s o -o c c l u s i ve events that result in acute pain cri s e s, with chronic and pro g re s s i ve tissue damage 7.
c a r riers have an increased risk of art e rial va s-cular disease and venous thrombosis 9.
The high turn over of red blood cells (RBC) o b s e rved in sickle-cell patients is associated with lower serum levels of folate and vitamin B12 1 0, leading to the increased tHcy serum or
plasma levels that have been implicated in va s-cular endothelium injury 1 1. Even in the
ab-sence of genetic predisposition or tra d i t i o n a l risk factors such as C-re a c t i ve protein and MTHFR, the increased tHcy levels have been c o n s i d e red an important predictor of mort a l i t y in patients with angiographically defined coro-n a ry art e ry disease 1 2.
Bahia, a State in No rtheast Brazil, has a high interethnic admixture. The strong Afri c a n gene pool 1 3has resulted in a high fre q u e n c y
of hemoglobinopathies, with a pre valence of S h e te rozygotes (AS) reaching 14% in groups of A f rican descendants 1 4. In this article we re p o rt
on a molecular chara c t e rization of the C677T MTHFR gene polymorphism in a newborn p o p-ulation from two different maternity hospitals in Sa l va d o r, investigating its association with the presence of va riant hemoglobins.
Material and methods
S u b j e c t s
A total of 843 newborns we re studied. After ob-taining approval from the Re s e a rch Et h i c s Committee of the Oswaldo Cruz Fo u n d a t i o n , we enrolled newborns from the Tsylla Ba l b i n o public maternity hospital and the Santo Amaro p ri vate maternity hospital. Although both are located in Sa l va d o r, the socioeconomic chara c-t e risc-tics of women giving birc-th ac-t c-these c-two hospitals are quite different. Sample collection o c c u r red from May to December 2000. Ei g h t h u n d red fort y- t h ree newborn babies we re s c reened for the C677T MTHFR gene polymor-phism and 763 for the hemoglobin pro f i l e.
Blood collection
Pe d i a t ricians and nurse staff collected a cord -blood sample from each infant, using EDTA anticoagulant. Samples we re re f ri g e rated at 4oC for a maximum of eight hours before the
hemoglobin profile inve s t i g a t i o n .
Hemoglobin analysis
Hemoglobin analyses we re perf o rmed by c a t i o n -e xchane high p-erf o rmanc-e liquid chro m a t o g-raphy (HPLC) (Bi o Rad Va ri a n t®, Ca l i f o rn i a ,
USA), according to the manufacture r’s instru c-t i o n s.
DNA extraction
DNA was isolated from cord-blood leukocytes using the GFX®Genomic Blood DNA Pu ri f i c
a-tion KIT (Amersham Ph a rmacia Biotech In c. , P i s c a t a w a y, USA), following the manufacture r’s i n s t ru c t i o n s.
C677T MTHFR gene polymorphism
C677T MTHFR gene polymorphism was inve s-tigated by PCR, as previously described by Frosst et al. 1. The generated 198 bp PCR pro
d-uct was digested by the Hi nf I re s t riction en-zyme (New England BioLabs In c., Tozer Ro a d , Be ve r l y, MA, USA), resulting in 175 and 23 bp f ragments in the homozygous T state, and 198, 175, and 23 bp fragments in heteroz y g o t e s. T h e wild type remains undigested, pre s e rving the o riginal 198 bp fra g m e n t .
R e s u l t s
The total T-allele frequency was 0.23, with a p re valence of 36.2 and 5.3% for the C/T and T / T g e n o t y p e s, re s p e c t i ve l y. Howe ve r, the allele fre-quency was higher among newborns of the Santo Amaro maternity hospital than from the n e w b o rns of the Tsylla Balbino maternity hos-pital, with a statistically significant differe n c e f rom the T/T genotype distribution (Table 1).
As shown in Table 2, out of 763 newborn s, 60 we re FAS heterozygotes (53 from Tsylla Ba l-bino and seven from Santo Amaro); 30 we re FAC heterozygotes (27 from Tsylla Balbino and t h ree from Santo Amaro); one was FC, six we re FSC, and one was FS (all from Tsylla Ba l b i n o ) . The frequency of va riant hemoglobins was higher at the Tsylla Balbino than the Sa n t o A m a ro maternity hospital, with a statistically significant difference for FAS, χ2= 14.14 and p
value < 0.001, and for FAC genotype distri b u-t i o n s, χ2= 7.80 and pvalue = 0.005. The
ob-s e rved relationob-ship between the C677T MTH-FR gene polymorphism and different hemoglo-bin patterns was established in a total of 683 samples (Table 3).
D i s c u s s i o n
l ower frequency among Africans 1 5. Krieger et
al. 1 3estimated in a sample from No rt h e a s t
Brazil that it was some 97% of the way to in-t e rein-thnic panmixia. An inin-tensive African slave t rade occurred from the 16t hthe 19t hc e n t u ri e s 1 6, and more than five million Africans of
sev-e ral nationalitisev-es sev-entsev-ersev-ed Brazil bsev-etwsev-esev-en 1850 and 1950 1 7. In Brazil, Arruda et al. 1 8found a
T-allele frequency of 10% among people of Eu ro-pean descent; 1.45% among Blacks; and 1.2% among In d i a n s. Pe rez et al. 1 9, studying
chil-d ren with spina bifichil-da anchil-d contro l s, founchil-d p re valence rates for the T/T genotype of 7.64 and 10.32%, re s p e c t i ve l y. The T-allele fre q u e n-cy of 0.23 and the pre valence of C/T and T / T genotypes found in this re p o rt we re higher than those observed in other studies of Afri c a n and African-descent populations worldwide
2 0 , 2 1 , 2 2and lower than those found in studies of
Eu ropeans 2 3. This result was probably
influ-enced by the high rate of racial admixture in the Bahian population. The T/T genotype dis-p l a yed a statistically significant difference be-t ween be-the groups of newborns from be-the be-two m a t e rnity hospitals, with a lower frequency at the Tsylla Balbino maternity Hospital. T h e mothers who gave birth at the Santo Amaro m a t e rnity hospital are pri m a rily re p re s e n t a t i ve of the Eu ropean-descendent portion of the Bahian population, confirming the higher T- a l-lele frequencies previously described in this continental group 2 4.
In contrast to the C677T MTHFR gene poly-morphism distribution, the va riant hemoglo-bin frequencies we re higher at the Tsylla Ba l b i-no than at the Santo Amaro maternity hospital. These differences are probably due to the eth-nic composition of the newborns from the two h o s p i t a l s. Az ê vedo et al. 1 4s h owed an incre a s e
in va riant hemoglobin allele frequencies in Eu-ropean-descendent Ba h i a n s, which we re not re s t ricted to African-descendent gro u p s, but with intermediate numbers among mulattos.
The frequencies of C/T or T/T genotypes among FAS newborns we re 35.7% and 5.4%, re-s p e c t i ve l y. The T-allele prere-sence hare-s been are-sre-so- asso-ciated with increased tHcy serum leve l s. Kha-j u ria & Houston 2 5s h ow in vitro evidence of a
d i rect mechanism by which homocysteine, at physiologically and pathophysiologically re l e-vant concentra t i o n s, may induce thro m b o s i s.
Even though sickle cell patients ro u t i n e l y using folic acid to improve ery t h ro p o i e s i s, fo-late status can be found at sub-clinical leve l s when compared to Hb AA normal contro l s e rum folate levels 1 0. Lowenthal et al. 2 6h
y-p o t h e s i zed that the concentration of folate re-q u i red to norm a l i ze plasma homocysteine
lev-Table 3
The C677T MTHFR polymorphism and hemoglobin patterns among 683 n e w b o rns from the Tsylla Balbino and Santo Amaro maternity hospitals. S a l v a d o r, Bahia, Brazil, 2000.
Hemoglobin C677T MTHFR gene Polymorphism
p ro f i l e C/C (%) C/T (%) T/T (%) To t a l
FA 349 (59) 211 (35.6) 32 (5.4) 592
FAS 33 (58.9) 20 (35.7) 3 (5.4) 5 6
FAC 18 (66.7) 8 (29.6) 1 (3.7) 2 7
F C – 1 (100.0) – 1
F S C 4 (66.6) 1 (16.7) 1 (16.7) 6
F S – 1 (100.0) – 1
To t a l n= 404 n= 242 n= 37 n= 683
n= number of samples.
Table 1
P revalence of the C677T MTHFR gene polymorphism among 843 n e w b o rns from the Tsylla Balbino and Santo Amaro maternity hospitals. S a l v a d o r, Bahia, Brazil, 2000.
G e n o t y p e Tsylla Balbino (%) Santo Amaro (%) Total (%)
C / C 379 (61.0) 114 (51.4) 493 (58.5)
C / T 216 (34.8) 89 (40.1) 305 (36.2)
* T/T 26 (4.2) 19 (8.6) 45 (5.3)
To t a l n= 621 n= 222 n= 843
A l l e l e Allele fre q u e n c y Allele fre q u e n c y Allele fre q u e n c y
C 0 . 7 8 0 . 7 1 0 . 7 7
T 0 . 2 2 0 . 2 9 0 . 2 3
n= number of samples; * Diff e rences in T/T genotype, χ2= 8.08; p = 0.004.
Table 2
Hemoglobin profile distribution among newborns from the Tsylla Balbino and Santo Amaro maternity hospitals. Salvador, Bahia, Brazil, 2000.
Hemoglobin Tsylla Balbino (%) Santo Amaro (%) Total (%) p a t t e rn
FA 421 (82.7) 244 (96) 665 (87.2)
FA S * 53 (10.4) 7 (2.8) 60 (7.9)
FA C * * 27 (5.3) 3 (1.2) 30 (3.9)
F C 1 (0.2) – 1 (0.1)
F S C 6 (1.2) – 6 (0.8)
F S 1 (0.2) – 1 (0.1)
To t a l n = 509 n = 254 n = 763
n= number of samples; *χ2= 14.14 and p value < 0.001;
els in patients with sickle cell disease may be higher than that of normal contro l s, and that patients with sickle cell disease have a higher n u t ritional re q u i rement for folic acid than the g e n e ral population. These facts can contri b u t e to raising tHcy serum leve l s, described as a ri s k factor for coro n a ry art e ry disease 2 7. The T- a
l-lele presence found in the newborns in this study could provide an important basis for further studies focusing on potential risks of va s o -o c c l u s i ve events in these individuals.
The Bahia State Foundation for He m a t o l o g y and Transfusion T h e ra py (HEMOBA) has re g i
s-t e red 942 SS pas-tiens-ts, and das-ta from s-the neona-tal screening pro g ram for va riant hemoglobins f rom APAE (the Association of Pa rents and Friends of Children with Disabilities) in Sa l-va d o r, Bahia re p o rts one SS for eve ry 423 new-b o rn s. Blood samples from these SS patients and newborns can be re f e r red to specialize d l a b o ra t o ries to perf o rm molecular inve s t i g a t i o n of the C677T polymorphism in the MTHFR g e n e. In our opinion, polymorphisms in genes responsible for folate and homocysteine meta-bolic pathways should be investigated in Ba h i a and other SS populations worldwide.
A c k n o w l e d g e m e n t s
We wish to thank the dire c t o r s, physicians, and nurs-es from the Tsylla Balbino and Santo Amaro matern i-ty hospitals for their assistance in cord-blood collec-tion. We are grateful to Craig Mi l roy for an English re-view and the Bahia State Ce n t ral Labora t o ry for pro-viding all the equipment necessary for biochemical a n a l y s e s.
C o n t r i b u t o r s
F. D. Couto participated in the blood sample collec-tion in the hospitals, hemoglobin analysis, DNA ex-t racex-tion, molecular analyses, and drafex-ting of ex-the m a n u s c ript. E. V. Ad o rn o, J. F. Me n eze s, and J. P. Mo u ra Neto participated in the blood sample collec-tion in the hospitals, hemoglobin analysis, and DNA e x t raction. M. Rego participated in the statistical a n a l y s i s. M. G. Reis participated in the interpre t a t i o n of the results and drafting and revision of the manu-s c ript. M. S. Go n ç a l vemanu-s manu-supervimanu-sed the labora t o ry a n a l y s e s, interpretation of the re s u l t s, and dra f t i n g and revision of the manuscri p t .
R e s u m o
O polimorfismo C677T no gene da MTHFR tem sido associado ao aumento dos níveis séricos de homocis-teína.total (tHcy), descrito como fator de risco para o d e s e n volvimento de doenças card i ova s c u l a re s .O i t o-centos e quarenta e três recém-nascidos (RNs), de duas maternidades difere n t e s , uma pública e a outra priva-d a , em Salva priva-d o r, Ba h i a , Brasil foram triapriva-dos para o p o l i m o rfismo C677T por PCR e RFLP. A freqüência do alelo T foi de 0,23 e as pre valências dos genótipos C/T e T/T foram de 36,2% e 5,3%, re s p e c t i va m e n t e . A fre-qüência do alelo T diferiu e a pre valência do genótipo T/T foi mais elevada entre os RNs da maternidade pri-va d a . O perfil de hemoglobinas (Hb) foi determinado por HPLC em 763 RNs. A freqüência de Hbs va r i a n t e s foi mais elevada entre os RNs da maternidade pública Tsylla Balbino do que na maternidade privada do Hospital Santo Am a ro. A associação do polimorf i s m o C677T e o perfil de Hbs foram estudados em 683 RNs, a p resentando freqüência elevada da coexistência do alelo T e Hb va r i a n t e s . Estes resultados podem ser uti-lizados como base para estudos futuros sobre riscos p o-tenciais de eventos va s o - o c l u s i vos nestes indivíduos.
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Submitted on 28/Ma y / 2 0 0 3
