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diagnosis since a female fetus 6ill either !e unaffected or an asymptomatic carrier of the mutation. >n the other hand, if

a male fetus is diagnosed, there is a 50 percent chance he has the disease so in asi e testing for prenatal diagnosis

should !e offered.

Ae+ determination is also useful in the management of pregnancies in 6hich the fetus is at ris' for congenital adrenal

hyperplasia. ffected female fetuses can de elop genital am!iguityDgenital iriliEation, 6hich can !e pre ented !yadministering de+amethasone to the mother throughout pregnancy. *f the fetus is 'no6n to !e male, steroid therapy can

!e omitted and its associated side effects and complications can !e a oided. $Aee /"reatment of classic congenital

adrenal hyperplasia due to 21%hydro+ylase deficiency in infants and children/, section on <?renatal therapy< .&

systematic re ie6 and meta%analysis of nonin asi e fetal se+ determination using cell%free fetal found that real%

time #uantitati e $ "F&%?C outperformed con entional ?C $ "F%?C sensiti ity and specificityB 97 and 99 percent,

respecti ely con entional ?C sensiti ity and specificityB 94 and 9:.3 percent, respecti ely& 33 -. Aensiti ity increased

6ith ad ancing gestational ageB

?rior to : 6ee's $sensiti ity :4.5 percent, specificity 99.1 percent&

: to 12 6ee's $sensiti ity 94.8 percent, specificity 98.9 percent&

13 to 20 6ee's $sensiti ity 95.5 percent, specificity 99.1 percent&

fter 20 6ee's $sensiti ity 99.0 percent, specificity 99.7 percent&

"ests on urine 6ere unrelia!le.

irect%to%consumer mar'eting ia the internet and print media of fetal se+ testing ser ices has made tests for fetal se+

determination 6idely a aila!le, although the accuracy of tests from unregulated la!oratories is #uestiona!le 34 -, and

their a aila!ility raises important ethical concerns.

Rhesus t ping — "he same approach is used for determining fetal h$ & status in h$ &%negati e 6omen. nonin asi e test for fetal h$ & in the maternal circulation is commercially a aila!le in the Gnited Atates, and is

6idely used in =urope to reduce the need for unnecessary fetal sur eillance in isoimmuniEed h$ &%negati e 6omen

carrying an h$ &%negati e fetus 35,37 -. $Aee /Management of pregnancy complicated !y hesus $ h&

alloimmuniEation/, section on <Cell free fetal testing to determine fetal h$ & type< .&

nother application is a oidance of unnecessary prenatal immunoglo!ulin in(ections for pre ention of isoimmuniEation in

h$ &%negati e 6omen carrying h$ &%negati e fetuses 3: -. "he cost effecti eness of this approach has not !een

determined.

+ingle gene disorders — "he nonin asi e prenatal diagnosis of single gene disorders is limited to detection of

paternally inherited mutations, 6hich 6ould not !e present in the maternal genome $assuming the mother is unaffected&.

@o6e er, techni#ues under study that measure the <relati e mutation dosage< of a particular gene in all cell%free in

the maternal !loodstream may help o ercome this limitation !y assessing the fetal allelic contri!ution to the maternal

pool 38 -.

)or autosomal dominant conditions, diagnosis depends on the identification of the paternal allele. Hhen the father is

affected, a /positi e/ test in maternal !lood signifies the presence of the defecti e paternal allele and an affected fetus

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$assuming the mother is unaffected&. ?aternally%inherited autosomal dominant diseases that ha e !een detected using

fetal in maternal !lood include @untington disease 39,40 -, myotonic dystrophy 41 -, and achondroplasia 42 -.

Hhen the mother is affected !y an autosomal dominant disease, nonin asi e fetal diagnosis is not possi!le !ecause

fetal nucleic acids deri ed from maternal alleles cannot !e distinguished from maternal nucleic acids.

*n the case of autosomal recessi e conditions 6here the parents are carriers of different mutations, a /negati e/ test for

the paternal mutation rules out an affected fetus. /positi e/ test indicates that the fetus has inherited the paternal

allele carrying the mutation ho6e er, the test cannot distinguish the fetus that is an unaffected carrier of the paternal

allele from the fetus that carries the paternal allele as 6ell as a maternal allele, and therefore is affected.

!ommercial availabilit — "here is less commercial interest in de eloping tests for fetal genetic disorders using cell%

free nucleic acids in maternal circulation than there is for diagnosing aneuploidy, since far more couples desire

aneuploidy testing than testing for Mendelian disorders. *t is unclear 6hether nonin asi e tests for common single gene

disorders such as cystic fi!rosis or !eta thalassemia 6ill !e commercially a aila!le in the ne+t fe6 years, although many

o!ser ers thin' that their de elopment is ine ita!le.

,etal aneuploidies — > erall, fetal le els in the maternal circulation are increased in trisomy 21 43,44 - and

trisomy 13 45 -, !ut not trisomy 18 45 -. "he challenge in detecting fetal aneuploidies is to identify an e+tra $or missing&

copy of a fetal chromosome in the maternal !lood 6here fetal constitutes only a small fraction of the cell%free .

"he amount of chromosome 21, for e+ample, cannot !e simply #uantified, !ecause a 50 percent increase of fetal

chromosome 21 in trisomy 21 $ o6n syndrome& 6ould !e lost in the /noise/ of !ac'ground maternal chromosome 21 in

the circulation 43 -.

ecause fetal gets lost among the more a!undant maternal , an alternati e strategy is to search for imprinted

genes 6ith m transcripts e+pressed only !y placental chromosome 21. "he se#uence ?I C4 in m is used for

this purpose, and a ratio is measured !et6een the #uantity of different alleles of the ?I C4 m in circulation. *t isassumed that the fetus carries a chromosome 21 from each parent and, therefore, $normal& disomy 21 should generate

e#ual amounts of t6o alleles of ?I C4 m . 2B1 or 1B2 ratio suggests trisomy. "his approach identified 9 of 10

cases of o6n syndrome and e+cluded 55 of 5: normal controls in one study 47 -.

limitation of this approach is that it only 6or's for fetuses that are heteroEygous for the single nucleotide

polymorphism $A ?& that is tested. fetus homoEygous for the A ? 6ould generate no ratio $all the A ?s are the

same& and therefore a diagnosis could not !e made. Hith the disco ery of more A ?s for imprinted genes on

chromosome 21, this approach may !e useful to a !roader proportion of the population.

nother promising approach le erages the difference in methylation !et6een maternal and fetal genes. "he

A= ?* 5 gene coding for maspin is hypomethylated in the placenta, !ut hypermethylated in maternal !lood cells. "hisgene resides on chromosome 18 therefore, an analysis that compares allele ratios among hypomethylated ersions of

the gene may distinguish the euploid fetus from the fetus 6ith trisomy 18 28,4: -. "here is some e idence that the same

approach may 6or' 6ith differentially%methylated genes on chromosome 21 29 -.

igital ?C / is !eing used to count the num!er of fetal chromosomes 21 of each allele, and therefore can !e used to

compare the ratio of fetal alleles and diagnose o6n syndrome $or other trisomies and monosomies& in heteroEygous

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fetuses. s pre iously mentioned, this technology is too resource intensi e at this time to !e cost%effecti e as a

commercial test 48 -.

"he use of chromosome%selecti e se#uencing $digital analysis of selected regions A - assay 6ith a fetal fraction

optimiEed ris' of trisomy e aluation )> "=- algorithm& is another promising e ol ing technology that has !een applied

to detection of trisomy 21 and 18 49%51-. *n a high%ris' population sampled in the first trimester, the sensiti ity of thistest for detecting trisomy 21 6as 100 percent $81D81 cases& the sensiti ity for trisomy 18 6as 9:.4 percent $3:D38

cases&, and the specificity for trisomy 21 and 18 6as 99.9: percent $288:D2888 cases& and 99.93 percent $2887D2888

cases&, respecti ely, at a cut%off of 1 in 100 51 -.

"he most relia!le a aila!le technology is massi ely%parallel se#uencing, a techni#ue that se#uences short segments of

cell%free in the maternal serum and computes a distri!ution of , !oth fetal and maternal, in the maternal serum

15,2: -. series of alidation studies using this techni#ue ha e demonstrated high sensiti ity for diagnosis of o6n

syndrome $sensiti ity :9 to 100 percent, 6ith false positi e rates !elo6 1 percent& 15,17,52%55 -, trisomy 18 $sensiti ity

9: to 100 percent& 53%55-, and trisomy 13 $sensiti ity :5 to :9 percent& 54,55 -. lthough there is some e idence that

this techni#ue can !e used to identify other less common aneuploidies and mosaicisms 6ith similarly high sensiti ity53%55-, more research is needed to pro e this capacity. $Aee /> er ie6 of prenatal screening and diagnosis of o6n

syndrome/, section on <Aecondary screening !y a maternal plasma !ased test for fetal < .&

!ommercial availabilit — onin asi e screening for aneuploidy using cell%free in the maternal !lood 6as made

a aila!le for clinical use in 2011 !y Ae#uenom $Aan iego, C &, and se eral other companies launched tests in 2012.

ecause of the small, !ut real, ris' of a false%positi e result, most e+perts ad ise using these assays as screening tests,

and confirming a positi e result 6ith in asi e prenatal diagnosis. )urthermore, !ecause most preliminary data came

from 6omen at high ris' for aneuploidy, the role of these assays in lo6%ris' populations is unclear.

Pregnanc complications — ltered le els of fetal nucleic acids in the maternal circulation are associated 6ith

pathologic conditions other than aneuploidy, !ut these conditions ha e !een the focus of fe6 studies. Circulating le els

of cell%free fetal nucleic acids are significantly ele ated in placental a!ruption 57 -, preeclampsia 5:%70-, preterm la!or

71 -, hyperemesis gra idarum 72 -, and polyhydramnios 73 -.

.ultiple gestation — "here is minimal information on cell%free le els in multiple gestations only a fe6 cases ha e

!een reported 53,74 -.

+ ..#R/ #ND R*!O..*ND#TION+

)ree fetal and can !e detected in the maternal circulation in the first trimester !oth are cleared

rapidly after deli ery. $Aee <Hhen are fetal cell%free nucleic acids found in maternal !loodK< a!o e.&

Most nonin asi e fetal testing is !ased upon detection of paternally inherited fetal alleles that are not present in

the maternal genome. )etal nucleic acids deri ed from maternal alleles cannot !e distinguished from maternal

nucleic acids. $Aee <?renatal diagnostic applications< a!o e.&

Gse of nonin asi e fetal h$ & testing in h$ &%negati e 6omen allo6s isoimmuniEed h$ &%negati e 6omen

to a oid unnecessary fetal sur eillance and allo6s unimmuniEed h$ &%negati e 6omen to a oid unnecessary

prenatal use of h$ & immune glo!ulin. nonin asi e test for fetal h$ & in the maternal circulation is

commercially a aila!le. $Aee < hesus typing< a!o e.&

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on%in asi e fetal se+ determination is a aila!le from academic and commercial la!oratories. "his information

is useful in fetuses at ris' for %lin'ed diseases or if there is a family history of congenital adrenal hyperplasia

$C @&. $Aee<)etal se+ determination< a!o e.&

on%in asi e fetal testing for single gene disorders of paternal origin is a aila!le from some academic

la!oratories. Gse of this technology is under e aluation, and until more data are a aila!le, reliance on these

tests may !e un6ise, particularly gi en the lo6 o erall ris' of in asi e testing. $Aee <Aingle gene disorders<

a!o e.&

"he role of nonin asi e testing for trisomy 21, trisomy 18 and trisomy 13 in clinical practice is e ol ing. Aome

clinicians are using non%in asi e testing as a screening test in high%ris' patients, particularly those 6ho 6ould

other6ise select in asi e diagnostic testing. $Aee <)etal aneuploidies< a!o e.&