9 6 4
CELL- FREE FETAL DNA I N MATERNAL PLASMA AND NONI NVASI VE PRENATAL DI AGNOSI S
Est er Silv eir a Ram os1
Ram os ES. Cell- fr ee fet al DNA in m at er nal plasm a and noninvasive pr enat al diagnosis. Rev Lat ino- am Enfer m agem 2 0 0 6 n ov em br o- dezem br o; 1 4 ( 6 ) : 9 6 4 - 7 .
The noninvasive nat ur e of t he det ect ion of fet al DNA in t he m at er nal cir culat ion r epr esent s t he gr eat est adv ant age ov er t he conv ent ional m et hods of pr enat al diagnosis. The applicat ions of t his m et hodology inv olv e
t he det ect ion of t he fet al sex , and diagnosis, int r a- ut er ine t r eat m ent , and evaluat ion of t he pr ognosis of m any diseases. Fet al cells det ect ed in t he m at er nal cir culat ion have also been show n t o be im plicat ed in aut oim m une
d iseases an d t o r ep r esen t a p ot en t ial sou r ce of st em cells. On t h e ot h er h an d , w it h t h e in t r od u ct ion of a t ech n ology t h at det ect s t h e fet al sex as ear ly as at 6 - 8 w eek s of gest at ion , t h er e is t h e possibilit y of ear ly
abor t ion based on sex select ion for social pur poses. This im plies an et hical discussion about t he quest ion. The in t r od u ct ion of n ew n on in v asiv e t ech n iq u es of p r en at al d iag n osis an d t h e k n ow led g e of t h e Nu r sin g Team r egar ding new m et hodologies can be of gr eat benefit t o t he m ot her and her childr en, and can help t he Genet ic
Counseling of t he fam ilies.
DESCRI PTORS: p r en at al d iag n osis; DNA; b lood ; st em cells; b lood g r ou p in com p at ib ilit y ; sex ; eclam p sia; epigen esis, gen et ic; et h ics
DNA FETAL LI BRE EN EL PLASMA MATERNO Y DI AGNÓSTI CO PRENATAL NO I NVASI VO
La nat ur aleza no inv asiv a de la inv est igación del DNA fet al en la cir culación m at er na r epr esent a una
vent aj a im por t ant e con r elación a los m ét odos convencionales de diagnóst ico pr enat al. El uso de est a m et odología im plica la det er m inación del sexo fet al y el diagnóst ico, el t r at am ient o int r a- út er o y la evaluación del pr onóst ico en m u ch as en f er m ed ad es. Las célu las f et ales d et ect ad as en la cir cu lación m at er n al t am b ién p u ed en ser
im plicadas en enfer m edades aut oinm unes y r epr esent ar una fuent e pot encial de células m adr e. Por ot r a par t e, con la in t r odu cción de u n a t ecn ología qu e det ect e el sex o f et al en t r e 6 - 8 sem an as de gest ación , ex ist e la
posibilidad de abor t o pr ecoz basada en la selección del sex o par a los pr opósit os sociales. Est o im plica u n a discu sión ét ica pr ev ia sobr e est e pr oblem a. La in t r odu cción de n u ev as t écn icas n o in v asiv as de diagn óst ico
pr enat al y el conocim ient o del Equipo de Enfer m er ía con r espect o a las nuev as m et odologías pueden ser m uy im por t ant es a la m adr e y a sus niños, y ay udar al Consej o Genét ico de las fam ilias.
DESCRI PTORES: diagn óst ico pr en at al; ADN; san gr e; célu las m adr e; in com pat ibilidad de gr u pos san gu ín eos; sex o; eclam psia epigén esis gen ét ica; ét ica
DNA LI VRE FETAL EM PLASMA MATERNO E DI AGNÓSTI CO PRÉ- NATAL NÃO I NVASI VO
A nat ur eza não inv asiv a par a o fet o da pesquisa de DNA fet al em cir culação m at er na r epr esent a sua
m aior v an t agem sobr e os m ét odos con v en cion ais de diagn óst ico pr é- n at al. As aplicações dest a m et odologia en v olv em a d et ecção d o sex o f et al e o d iag n óst ico, t r at am en t o in t r a- ú t er o e av aliação d o p r og n óst ico d e
v ár ias doen ças. Já as célu las f et ais det ect adas em cir cu lação m at er n a podem est ar en v olv idas em doen ças au t o- im u n es e r epr esen t ar u m a f on t e de célu las- t r on co par a as m ães. Com a in t r odu ção com er cial de u m a
t écnica que det ect a o sex o fet al ent r e 6- 8 sem anas, hav er ia o r isco de abor t os pr ecoces dev ido à seleção do sexo por pr opósit os sociais, t or nando necessár ia um a discussão pr évia sobr e os aspect os ét icos dest a quest ão. A in t r od u ção d e n ov as t écn icas n ão in v asiv as d e d iagn óst ico p r é- n at al e o con h ecim en t o d as m esm as p or
p ar t e d a Eq u ip e d e En f er m ag em p od er ão t r azer g r an d es b en ef ícios p ar a a m ãe e seu s f ilh os, b em com o au x iliar n o Acon selh am en t o Gen ét ico das fam ílias.
DESCRI TORES: diagnóst ico pr é- nat al; DNA; sangue; células- t r onco; incom pat ibilidade de gr upos sanguíneos;
sex o; eclam psia; epigên ese gen ét ica; ét ica
1 Physician w it h Residence in Clinical Genet ics, PhD in Genet ics, Pr ofessor School of Medicine of Ribeir ao Pr et o - Univer sit y of Sao Paulo, Br azil; e- m ail:
esram os@rge.fm rp.usp.br
Rev Lat ino- am Enfer m agem 2006 novem br o- dezem br o; 14( 6) : 964- 7 w w w . eer p. usp. br / r lae
Ar t igo de At ualização
9 6 5
A
ll pr egnant w om en hav e t he pot ent ial r isk t o p r esen t a n em b r y o w i t h a n o m a l i es. Th er ef o r e, m at ernal age should be t aken int o considerat ion in all pregnancies and fet al m orphologic ult rasonography and biochem ical t est s in m at er nal blood should alw ays be car r ied out( 1). How ev er, in special cases, t her e is t he need t o use invasive m et hods, such as chorionic villussam pling ( CVS) or am niocent esis, w hich r epr esent s a h ig h er r isk f or t h e em b r y o( 2 ). For t h is r eason , t h e t echnologies inv olv ing pr enat al diagnosis cont inue t o evolve, wit h em phasis on research for t he developm ent and im pr ov em ent of noninv asiv e t echniques.
F E T A L CE L L S I N T H E M A T E R N A L
CI RCULATI ON
The det ect ion of fet al cells in m at er nal lung p a r e n ch y m a a sso ci a t e d w i t h e cl a m p si a h a s b e e n descr ibed since t he 19t h cent ur y( 3), but t he pr esence of t hese cells in m at er nal per ipher al blood cont inues t o be t he subj ect of int ense research. Fet al cells appear
e a r l y i n t h e m a t e r n a l ci r cu l a t i o n d u r i n g t h e f i r st t r i m e st e r a n d co n t i n u e t o b e p r e se n t t h r o u g h o u t gest at ion. However, t he isolat ion of fet al cells from t he m at er nal cir culat ion is st ill t echnically com plex . Fet al cells can be pr esent in m at er nal blood at a r at io of
1: 100,000 m at ernal cells or less ( or approxim at ely one per m l of m at er n al blood in eu ploid pr egn an cies)( 4 ). Anot her pr oblem , in addit ion t o t heir sm all num ber s, is t he perm anence of fet al cells in m at ernal circulat ion
aft er t he pregnancy. There is a report of t he det ect ion of nuclear m ale DNA in a wom an who had her last son 27 years before t he st udy( 5). Ot her invest igat ions have shown t hat t he persist ent cells could have an im port ant r ole in w om en’s aut oim m une diseases. On t he ot her h a n d , t h e s e c e l l s a p p e a r t o h a v e s t e m c e l l
char act er ist ics, such as t he abilit y t o pr olifer at e and different iat e, which could be of benefit t o t he m ot hers( 4).
CELL- FR EE FET AL D N A I N M AT ER N AL
PLASMA
The quant it at ive analysis of t he fr ee fet al DNA dem on st r at ed t h at t h is can com pose u p t o 6 . 2 % of
t h e t o t a l D NA p r esen t i n t h e m a t er n a l p l a sm a( 6 ).
Apopt osis w ou ld par t ially ex plain t h e disappear an ce
of fet al DNA from m at ernal circulat ion aft er birt h, since t his is a fast r eact ion, concluded w it hin 2 t o 3 hour s aft er childbirt h. These result s ( a higher rat io in relat ion t o t he m at er nal m at er ial and a fast er disappear ance fr om t he m at er nal cir culat ion) dem onst r at e pr act ical su p er ior it y of t h e sear ch f or f et al DNA in m at er n al
p lasm a in r elat ion t o t h e p r ep ar at ion of f et al cells isolat ed fr om m at er nal w hole blood( 4, 7).
APPLI CATI ONS
M a n y g e n e t i c d i s e a s e s a r e c a u s e d b y m u t at ion s t h at r esu lt in su bt le dif f er en ces bet w een t h e seq u en ces of m at er n al an d f et al DNA, su ch as
ach on dr oplasia( 8 ) an d â- t h alassaem ia( 9 ). Man y ot h er c l i n i c a l a p p l i c a t i o n s , e s p e c i a l l y f o r s i n g l e g e n e disorders, have been described. We w ill discuss som e a p p l i ca t i o n s t h a t a r e i m p o r t a n t b e ca u se o f t h e i r incidence in t he populat ion.
Fet al Sex in g
Mo s t r e s e a r c h g r o u p s u s e s e q u e n c e s o f chr om osom e Y in m ale em br yos as a m ar ker of fet al DNA an d st an d ar d izat ion of t h e t ech n iq u es, d u e t o
t he fact t hat a nor m al w om an/ m ot her ( 4 6 , XX) does not possess t his chr om osom e in her genom e. Sex ing is also im port ant , m ainly for diseases w it h a recessive X- linked pat t ern of inherit ance, w it h girls being norm al
or being car r ier s of t he m ut at ion, but healt hy, w hile b oy s ar e n or m al or af f ect ed b y t h e d isease. Som e au t h o r s h av e u sed t h e p o l y m er ase ch ai n r eact i o n ( PCR) a n d a m p l i f i ca t i o n o f Y- sp e ci f i c se q u e n ce s, m a i n l y h i g h l y r e p e t i t i v e se q u e n ce s o r g e n e s l i k e
SRY( 10). Mor e r ecent ly, t he TSPY gene has also been
used in st udies of sm all DNA sam ples( 11- 12).
A n o t h e r a p p l i c a t i o n o f f e t a l s e x i n g i s Con gen it al Adr en al Hy per plasia ( CAH) , a disease of genet ic or igin w it h an aut osom al r ecessive pat t er n of i n h e r i t a n c e . Th e m o s t c o m m o n d e f e c t i s 2 1
-h y d r o x y l a se d e f i ci e n cy. Ho m o zy g o u s g i r l s f o r t -h e deficiency are born w it h m asculinizat ion of t he ext ernal g en it alia, m an y t im es r eq u ir in g su r g ical cor r ect ion . The affect ed boy s pr esent nor m al ex t er nal genit alia.
Pr en at al t r eat m en t of CAH w it h d ex am et h ason e t o Cell- fr ee fet al DNA in m at er nal...
Ram os ES.
9 6 6
p r e v e n t g e n i t a l a m b i g u i t y h a s b e e n su cce ssf u l l y u sed( 1 3 ). How ev er, t o m in im ize t h e side ef f ect s, t h e
int errupt ion of t herapy has been indicat ed in t he case o f a f f e ct e d o r n o r m a l m a l e e m b r y o s a n d n o r m a l
f e m a l e e m b r y o s. Fo r t h i s r e a so n , f e t a l se x i n g i s necessar y dur ing pr egnancy and is gener ally car r ied
ou t b y in v asiv e m et h od s. Non in v asiv e f et al sex in g b ased on f r ee f et al DNA in m at er n al p lasm a w ou ld
br ing t he addit ional advant age of ear ly discont inuat ion of m edicat ion in t he case of m ale em br y os( 10).
Rh alloim m u n izat ion
Rh a l l o i m m u n i za t i o n i s a v e r y i m p o r t a n t
pr oblem in m edical an d obst et r ical clin ical pr act ice, p o t e n t i a l l y l e a d i n g t o h e m o l y t i c d i s e a s e o f t h e
new bor n. For pr egnant negat iv e Rh pat ient s ( 15% of t he populat ion) , a posit ive Rh em br yo involves a 16%
r isk of sen sit izat ion t o t h e Rh an t ig en . Diag n ost ic pr ocedu r es an d in v asiv e t h er apy m ay be n ecessar y
t o r e d u c e p e r i n a t a l m o r t a l i t y o f p o s i t i v e Rh em b r y os( 1 4 ). Th u s, t h e ear ly d et ect ion of f et al Rh D
st at u s t h r ou gh f et al DNA in t h e plasm a of n egat iv e Rh m ot hers is of great im por t ance in defining t he need
for int ervent ions, w it h know n risks of gest at ional loss, or of gest at ion al im m u n opr oph y lax is.
Pr e- ecl am p si a
Pre- eclam psia is char act er ized by an incr ease
in ar t er ial p r essu r e an d p r ot ein u r ia af t er t h e 2 0 t h w eek of pr egnancy. I t s pot ent ial com plicat ions, such
a s e cl a m p si a , r e p r e se n t o n e o f t h e m o st se r i o u s pr oblem s for m at er nal and fet al healt h. Accor ding t o
som e aut hors, t here is an increase of fet al DNA in t he b lood of w om en w it h p r e- eclam p sia in com p ar ison
w i t h co n t r o l g r o u p s. Th e i n cr ea se i n t h e r a t es o f cir culat ing fet al and m at er nal DNA w ould cor r espond
t o t he degree of severit y of t he illness and, t herefore, t he lev el of fet al DNA m ay ser v e as a m ar k er of t he
p r o g n o si s an d sev er i t y o f t h e cl i n i cal p i ct u r e( 7 , 1 5 ). Alt hough m ost r esear cher s use t he Y- chr om osom e in
t h is sp ecif ic ap p licat ion , ot h er n on - g en d er m ar k er s h av e been st u died, in clu din g epigen et ic m ar k er s, t o
im pr ov e t h e n u m ber of pr egn an t w om en t h at cou ld be subm it t ed t o quant it at iv e inv est igat ion( 16).
DI SCUSSI ON AND CONCLUSI ON
Due t o t he significant risk of invasive prenat al d i ag n osi s, t h er e h as b een an i n t en si v e sear ch f or n on in v asiv e t ech n iq u es of f et al DNA sam p lin g . Th e det ect ion of fet al cells in t he m at ernal circulat ion has show n im plicat ions in aut oim m une diseases and t heir pot ent ial as a sour ce of st em cells for t he m ot her s.
Ho w ev er, t h e m et h o d o l o g y av ai l ab l e t o sear ch f o r t h ese cel l s i s ex p en si v e an d co m p l ex , d u e t o t h e per m anence of fet al cells in t he m at er nal cir culat ion af t er pr egn an cy an d t h eir scar cit y. I n con t r ast , t h e isolat ion of cell- fr ee fet al DNA fr om m at er nal plasm a is r elat iv ely easy and inex pensiv e and allow s for t he sim u lt an eou s pr ocessin g of m an y sam ples.
Th e t ech n iqu e of f et al sex in g f r om m at er n al b lood can b e u sef u l f or t h e st an d ar d izat ion of t h e m e t h o d o l o g y a n d i n c a s e s o f d i s e a s e s w i t h a r ecessiv e X- lin k ed pat t er n of in h er it an ce, w h er e on ly boy s w ou ld be af f ect ed. I n t r au t er in e det ect ion cou ld a l so l ea d t o a n ea r l y a n d o p t i m i zed t r ea t m en t o f d iseases in t h is g r ou p an d of ot h er illn esses su ch a s CA H . O n t h e o t h e r h a n d , o n e p r o b l e m i n in t r od u cin g a t ech n olog y t h at d et ect s f et al sex as ear ly as 6 - 8 w eek s of gest at ion is t h e possibilit y of abor t ion based on sex select ion f or social pu r poses. T h i s m a y o c c u r d u e t o t h e p o s s i b l e g r e a t e r p sy ch olog ical an d m or al accep t ab ilit y of an ear lier
i n t e r r u p t i o n o f p r e g n a n cy. I n Br a zi l , a co u p l e o f ser v i ces p er f o r m f et al sex i n g co m m er ci al l y, u si n g m at er n al b l ood . Th i s i m p l i es an et h i cal d i scu ssi on ab ou t t h e q u est ion .
Th e ear ly det ect ion of f et al Rh D st at u s w ou ld b e h i g h l y u s e f u l i n g e s t a t i o n s o f n e g a t i v e Rh p at ien t s. Besid es t h e im p or t an ce of t h e q u alit at iv e m e t h o d s f o r m a n y o t h e r g e n e t i c d i s e a s e s , t h e qu an t ificat ion of fet al DNA in t h e m at er n al cir cu lat ion seem s t o be a pot en t ial m ar k er of pr ogn osis in pr e-e cl a m p si a .
As descr ibed by anot her gr oup, t he pr egnant w om en m ay b e in t er v iew ed an d t h e sam p le b lood ca n b e t a k e n i n t h e i r h o m e s b y a n u r se( 1 7 ). Th e int roduct ion of new noninvasive t echniques of prenat al d iag n osis an d t h e n u r sin g t eam ’s k n ow led g e ab ou t new m et hodologies for pat ient or ient at ion can be of gr eat benefit t o m ot her s and childr en, and can help t he genet ic counseling of fam ilies.
Cell- fr ee fet al DNA in m at er nal...
Ram os ES.
9 6 7
REFERENCES
1 . Ci ce r o S, Sa cch i n i C, Re m b o u sk o s G, Ni co l a i d e s KH. Sonogr aphic m ar ker s of fet al aneuploidy - a r eview . Placent a 2 0 0 3 ; 2 4 ( 2 ) : S8 8 - S9 8 .
2 . Seeds JW. Diagn ost ic m id t r im est er am n iocen t esis: h ow saf e? Am J Ob st et Gy n ecol 2 0 0 4 ; 1 9 1 ( 2 ) : 6 0 7 - 1 5 . 3. At t w ood HD, Par k WW. Em bolism t o t he lungs by t r ophoblast . Br J Ob st et Gy n aecol 1 9 6 1 ; 6 8 : 6 1 1 - 7 .
4. Bianchi DW. Fet om at er nal cell t raffic, pr egnancy- associat ed p r og en it or cells, an d au t oim m u n e d isease. Best Pr act Res Clin Ob st et Gy n aecol. 2 0 0 4 ; 1 8 ( 6 ) : 9 5 9 - 7 5 .
5 . Bian ch i DW, Z ick w olf GK, Weil GJ, Sy lvest er S, DeMar ia MA. Male fet al pr ogenit or cells per sist in m at er nal blood for as lon g as 2 7 y ear s p ost p ar t u m . Pr oc Nat l Acad Sci USA 1 9 9 6 ; 9 3 ( 2 ) : 7 0 5 - 8 .
6. Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM et al. Quant it at ive analysis of fet al DNA in m at er nal plasm a and ser um : im plicat ions for noninv asiv e pr enat al diagnosis. Am J Hu m Gen et 1 9 9 8 ; 6 2 ( 4 ) : 7 6 8 - 7 5
7. Bischoff FZ, Lew is DE, Sim pson JL. Cell- fr ee fet al DNA in m at er nal blood: k inet ics, sour ce and st r uct ur e. Hum Repr od Up d at e 2 0 0 5 ; 1 1 ( 1 ) : 5 9 - 6 7 .
8. Sait o H, Sek izaw a A, Mor im ot o T, Suzuk i M, Yanaihar a T. Pr e n a t a l D NA d i a g n o si s o f a si n g l e g e n e d i so r d e r f r o m m at er n al p lasm a. Lan cet 2 0 0 0 ; 3 5 6 : 1 1 7 0 .
9. Chiu RW, Lau TK, Leung TN, Chow KC, Chui DH, Lo YM. Pr en at al ex clu sion of â- t h alassaem ia m aj or by ex am in at ion of m at er n al p lasm a. Lan cet 2 0 0 2 ; 3 6 0 : 9 9 8 - 1 0 0 0 . 1 0 . Rij n der s RJ, v an der Sch oot CE, Bosser s B, de Vr oede MA, Ch r ist iaen s GC. Fet al sex det er m in at ion fr om m at er n al p l a s m a i n p r e g n a n c i e s a t r i s k f o r c o n g e n i t a l a d r e n a l h y p er p lasia. Ob st et Gy n ecol 2 0 0 1 ; 9 8 ( 3 ) : 3 7 4 - 8 .
1 1 . Pier ce KE, Rice JE, San ch ez JA, Br en n er C, Wan gh LJ. Real- t im e PCR using m olecular beacons for accur at e det ect ion of t he Y chr om osom e in single hum an blast om er es. Mol Hum Rep r o d 2 0 0 0 ; 6 ( 1 2 ) : 1 1 5 5 - 6 4 .
12. Bar t m ann AK, Caet ano LC, Rios AF, Vila RA, Ram os ES. TSPY det ect ion in blood, bu ccal, an d u r in e cells of pat ien t s w it h 45,X karyot ype. Am J Med Genet 2004; 130A( 3) : 320- 1. 1 3 . New MI . An Updat e of Con gen it al Adr en al Hy per plasia. An n N Y Acad Sci. 2 0 0 4 ; 1 0 3 8 : 1 4 - 4 3 .
14. Lo YM. Fet al RhD genot yping fr om m at er nal plasm a. Ann Med 1 9 9 9 ; 3 1 ( 5 ) : 3 0 8 - 1 2 .
1 5 . Z h on g XY, Holzg r ev e W, Hah n S. Cir cu lat or y f et al an d m at er n al DNA in pr egn an cies at r isk an d t h ose affect ed by p r eeclam p sia. An n N Y Acad Sci 2 0 0 1 ; 9 4 5 : 1 3 8 - 4 0 . 16. Poon LL, Leung TN, Lau TK, Chow KC, Lo YM. Differ ent ial DNA m et hylat ion bet w een fet us and m ot her as a st r at egy for d et ect in g f et al DNA in m at er n al p lasm a. Clin Ch em 2 0 0 2 ; 4 8 ( 1 ) : 3 5 - 4 1 .
1 7 . Br en n an P, Bar r et t J, Fiddler M, Th om son W, Pay t on T, Silm an A. Mat er nal- fet al HLA incom pat ibilit y and t he cour se of i n f l am m at or y ar t h r i t i s d u r i n g p r eg n an cy. J Rh eu m at ol . 2 0 0 0 ; 2 7 ( 1 2 ) : 2 8 4 3 - 8 .
Recebido em : 10.10.2005 Apr ovado em : 13.9.2006
Cell- fr ee fet al DNA in m at er nal...
Ram os ES.
