FOCUSED ISSUE OF THIS MONTH J Korean Med Assoc 2015 November; 58(11): 995-1002 pissn 1975-8456 / eissn 2093-5951 http://dx.doi.org/10.5124/jkma.2015.58.11.995 모체혈장내유리 DNA 를이용한비침습적산전검사 김건우 함춘여성크리닉 Non-invasive prenatal test using cell free DNA Kunwoo Kim, MD Hamchoon Women s Clinic, Seoul, Korea Although conventional prenatal screening tests for Down syndrome have been developed over the past 20 years, the positive predictive value of these tests is around 5%. Through these tests, many pregnant women have taken invasive tests including chorionic villi sampling and amniocentesis for confirming Down syndrome. Invasive test carries the risk of fetal loss at a low but significant rate. There is a large amount of evidence that non-invasive prenatal test (NIPT) using cell free DNA in maternal serum is more sensitive and specific than conventional maternal serum and/or ultrasound screening. Therefore implementing NIPT will increase aneuploidy detection rate and concurrently decrease fetal loss rate accompanying invasive test. More than 1,000,000 NIPT were performed globally since 2011. The uptake rate of NIPT is expected to increase more rapidly in the future. Moreover, as a molecular genetic technique advances, NIPT can be used for not only common aneuploidy screening but single gene disorder, microdeletion, and whole fetal genome sequencing. In this review, I will focus on the NIPT for common aneuploidies such as trisomy 13, 18, and 21. Key Words: Down syndrome; Maternal serum screening tests; Cell free DNA; Non-invasive prenatal test 서론 다운증후군은전세계적으로약 1,000 명출생당한명 정도태어나는것으로알려진가장흔한염색체이상으 로평생동안다양한의료적도움이필요한중요한질병 이다 [1]. 1980 년대부터다운증후군에대한출생전선별 검사는많은발전을거듭해오고있다. 산모의나이, 초 음파소견 (nuchal translucency), 모체혈액의단백질 (pregnancy associated plasma protein-a, human Received: September 3, 2015 Accepted: September 17, 2015 Corresponding author: Kunwoo Kim E-mail: mdkkw@hamchoon.com Korean Medical Association This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. chorionic gonadotropin 등 ) 등을이용한선별검사로약 5% 의위양성율로 90% 의다운증후군을선별해낼수있다 [2]. 그러나이런선별검사의가장큰문제는검사의양성예측도가 5% 정도로낮기때문에너무많은융모막검사나양수검사같은침습적인산전검사가행해지고, 이로인해태아가유산될수있다는것이다. 1997년임산부혈장에서 Y 염색체에서유래한 cell free DNA (cfdna) 가발견됨으로써 [3] 산전진단은새로운전기를마련하게되었다. 태반에서기원한 apoptotic trophoblasts로부터유래된 cfdna는약 150 bp 크기의잘려진 DNA 조각들이다 [4]. 임신 4주이후부터발견되기시작해서 10주이후에는평균 10% 정도가태반에서기원한 cfdna이고나머지 90% 는모체에서유래한 cfdna이다 [5-8]. 태반에서기원한 cfdna 양은개인에따라다양할수있는데, 일반적으로임신주수가증가할수록증가하고, 모체의체중이증가할수록감소한다 [9,10]. 태반에서기원한 cfdna 모체혈장내유리 DNA 를이용한비침습적산전검사 995
J Korean Med Assoc 2015 November; 58(11): 995-1002 Counting NIPT using cfdna Genotyping 된 cfdna 만을 sequ encing 하는 targeted massively parallel sequencing 으로분류할수있다 (Figure 1). S-MPS Sequenom (MaterniT21) Illumina (Verifi) BGI (China) (NIFTY) T-MPS Ariosa (Harmony) Figure 1. Classification of non-invasive prenatal test (NIPT). cfdna, cell free DNA; S-MPS, shotgun massively parallel sequencing; T-MPS, targeted massively parallel sequencing; SNP, single nucleotide polymorphism; BGI, Beijing Genomics institute. 는분만 2 시간이내모체혈장에서발견되지않기때문에 산전진단의좋은재료가될수있다 [11]. Sequencing 기 술과 bioinformatics 의발달로검사비용이줄어듦에따 라 2011 년부터 cfdna 를이용한다운증후군과같은흔 한염색체이상에대한선별검사의상업적서비스가중 국과미국에서처음시작되었다. 현재 cfdna 는염색체 이상에대한선별검사뿐만아니라 fetal whole genome sequencing [12,13], fetal methylome sequencing [14], fetal transcriptome sequencing [15] 에도이용될수있 게되었다. 본고찰에서는지금상업적으로시행이되고 있는 cfdna 를이용한다운증후군같은흔한염색체이상 에대한비침습적산전검사 (non-invasive prenatal test, NIPT) 에초점을맞추고자한다. SNP Natera (Panorama) 2. Principle of counting method 1) Shotgun massively parallel sequencing 모체혈장에존재하는모든 cfdna 조각을증폭하여 sequencing 을한후, 몇번염색체에서유래된 DNA 조각인지를분류하고 (alignment), 그개수를 counting한다. 다운증후군처럼 21번염색체가하나더있는경우에 21번에서유래된 DNA 조각이상대적으로조금더많을것이기때문에이차이를이용하여선별해낸다 [16,17]. 예를들어정상태아를임신한것을알고있는산모군의혈장에서 21번염색체에서유래한 DNA 조각이평균 1.5% 를차지하고표준편차가 0.02 인경우, 검사한샘플에서 21번염색체에서유래한 DNA 조각이 1.58% 를차지한다면 Z-s core=(1.58-1.5)/0.02=0.08/0.02=4이다. 일반적으로 Z-score가 3 이상인경우다운증후군고위험군으로분류되므로이경우다운증후군고위험군으로분류될수있다 (Figure 2). 2) Targeted massively parallel sequencing 비용을줄이고, 효율성을높이기위해선별하고자하는염색체 13, 18, 21, X, Y에서유래된 cfdna 조각만을선택적으로증폭하여 sequencing한후에 counting한다. 정상군과비교하여 trisomy나 monosomy를선별해낸다 [18]. Principle of Non-invasive Prenatal Test 1. Classification of non-invasive prenatal test 현재전세계적으로 NIPT 서비스를공급하고있는회사는 5개정도이고, 국내외의다른회사들에서도서비스를준비중이다. 각회사별로조금씩의차이는있지만분석방법에따라크게분류하면 counting method와 single nucleotide polymorphism (SNP) genotyping으로분류할수있고, counting method는모체혈장의모든 cfdna를 sequencing 하는 shotgun massively parallel sequencing 과선별대상이되는염색체 13, 18, 21, 성염색체에서유래 3. Principle of single nucleotide polymorphism method SNP는인간집단에서 1% 이상의빈도로존재하는단일염기서열의변이로정의되며, 유전체변이중가장흔한것으로 1억 4천만개이상이밝혀져있다 (dbsnp build 144). 모체혈액을원심분리하면혈장, buffy coat, 적혈구의세층으로분리가된다. 이중 buffy coat는모체백혈구들의집합으로이를분석할경우모체만의유전형을알수있다. 혈장 ( 모체 cfdna + 태반 cfdna) 과 buffy coat에대해서약 20,000개의 SNP site를 multiplex polymerase chain reaction으로증폭한후 sequencing한다. SNP 유전형과감 996 대한의사협회지
Kim K Maternal serum cell free DNA test for aneuploidy Amplification Sequencing AATCTGCC... AGGATCGC... TTAATCTG... Alignment Counting %chr.21= Unique count for chr 21 Total unique count In euploid %chr.21: mean = 1.5% SD = 0.02 Test sample %chr.21 = 1.58% Maternal cfdna Placental cfdna 1 2 3...21 X Y Z-score =(1.58-1.50)/0.02 = 4 (high risk call) Figure 2. Principle of single nucleotide polymorphism method. SD, standard deviation; cfdna, cell free DNA. 100% A Mother AA, fetus AA "Allele distribution" 0% A Mother AA, fetus AB Mother AB, fetus AA Mother AB, fetus AB Mother AB, fetus BB Mother BB, fetus AB Mother BB, fetus BB Disomy Trisomy Indivisual SNP positions Mother AA, fetus AAA Mother AA, fetus AAB Mother AB, fetus AAA Mother AB, fetus AAB Mother AB, fetus ABB Mother AB, fetus BBB Mother BB, fetus ABB Mother BB, fetus BBB Figure 3. Simplified principle of single nucleotide polymorphism (SNP) based non-invasive prenatal test. Each dot is one SNP and is the sum of both the maternal and fetal contribution. SNPs are biallelic and thus have only 1 of 2 DNA bases possible (which we reference as A and B). The SNPs are only 1 per location going left to right (like the chromosome was lying on its side), total about 3,000 SNPs per chromosome (you could see this if it were spread out more). Top of profile is 100% A 0% B. Continuing to go down the profile, A decreases and B increases until the bottom is 100% B and 0% A. RED SNPs are where mom is AA. The top line is where baby is also AA (thus 100% A). The second has 75% A and 25% B (baby AB). Green SNPs are where mom is AB. Again increasing B% from the 1st to third line. Blue SNPs are where mom is BB. Again increasing B% until the final line where baby and mom are 100% B. When the mother s SNP is heterozygous (Green SNP), an extra band appears when the fetus is trisomic. However, the band positions are dependent on allele frequency and fetal fraction. When the mother s SNP is homozygous (red and Blue SNP), one band is shifted when the fetus is trisomic. Amount of shift is dependent on allele frequency and fetal fraction. Not all SNP profile results are visually conclusive and thus why the algorithm is needed [20]. 양성율이낮기때문에양수검사와융모 막검사같은침습적인검사를줄일수있 다. 한보고에따르면기존의모체혈액 검사로다운증후군고위험군으로선별 된경우 NIPT 를통해 2 차적인선별을한 다면침습적인검사가 90% 이상줄어들 고, 침습적인검사에따른태아유산도 90% 이상감소할것으로기대된다 [23]. Limitation of Non-invasive Prenatal Test 1. False positive 실제태아는문제가없지만 NIPT 결 과는문제가있는것으로나오는경우이 수분열시의교차등을고려하여태아의유전형을추정하는 데, 정상, trisomy, monosomy 중가장가능성이높은상태 를유추함으로써선별해낸다 (Figure 3) [21,22]. 다. 흔한원인은다음과같다. 1) Confined placental mosaicism 태아의염색체는정상이지만태반에만비정상핵형을가 진세포가있는경우이다. 융모막검사결과의분석을보면 Performance of Non-invasive Prenatal Test 2008 년이후로 NIPT 의임상적효용성에대한많은연구 가있었다. 최근에발표된메타분석을보면기존의모체혈액 검사와초음파를이용한선별검사보다는훨씬뛰어난성적을 보여준다 (Table 1) [22,23]. 무엇보다다운증후군에대한위 약 1-2% 에서발견이된다 [24,25]. NIPT는태반에서유래된 trophoblast의 cfdna이므로 confined placental mosaicism 이있는경우 NIPT 결과는비정상으로나올수있다. 2) Vanishing twin 임신초기쌍태임신이었으나한명의태아만생존하는경우로일찍산전초음파가시행되지않는경우, 알기어려운경우가있다. 한보고에따르면 NIPT 검사에서위양성이나온경우약 15% 는 vanishing twin 때문이다 [26]. 997 대한의사협회지 모체혈장내유리 DNA 를이용한비침습적산전검사 997
J Korean Med Assoc 2015 November; 58(11): 995-1002 Table 1. Comparison of NIPT vs. standard screening test 3) Maternal mosaicism 특히 monosomy X 에대한선별검사에서중요하다. 여성 의나이가증가할수록 X 염색체소실현상이증가한다. 25 세 미만에서는 1% 미만이지만 25 세이상에서는나이에비례해 서 X 염색체소실현상이증가한다 [27]. NIPT 에서성염색체 이상으로나온경우약 8.56% 는모체성염색체모자이시즘 에의한위양성이다 [28]. 4) Occult maternal malignancy 125,456 건의 NIPT sample 을분석한결과 3,757 건에서 이상소견이발견되었고, 이중 10 건에서숨겨진산모의암 이발견되었다. 이중 7 건에서는두가지이상의 aneuploidy 소견이동시에관찰되었다 [29]. 이런경우태아는정상염색 체이나 NIPT 결과는비정상으로나올수있다. 5) Maternal copy number variation 모체 18 번염색체중복이 NIPT 위양성의원인으로보고 되었다 [30]. 2. False negative 실제태아는문제가있지만 NIPT 결과는문제가없는것 으로나오는경우이다. 기존의선별검사보다는그확률이매 우낮지만, 실제로보고가되고있다 [31]. False negative 의 주요한원인으로는모체 cfdna 과비교해서태반에서유래 한 cfdna 가너무작은경우이다 [32]. 3. Test failure NIPT [21] Standard screening test a) [22] Sensitivity FPR Sensitivity FPR Trisomy 21 99.2 0.09 78.9 5.4 Trisomy 18 96.3 0.12 80.0 0.31 Trisomy 13 91.7 0.11 50.0 0.25 Monosomy X 90.3 0.23 NA NA Values are presented as %. NIPT, non-invasive prenatal test; FPR, false positive rate; NA, not available. a) Standard screening test: maternal serum pregnancy associated plasma protein-a, beta human chorionic gonadotropin, and nuchal translucency. 서비스를제공하는회사에따라다르지만 0.8-8.1% 정 도에서는결과를내지못하는경우가존재한다 ( 성염색체이 상제외 )[21]. 검사결과를못내는경우그원인으로태반으 로부터유래한 cfdna 가 4% 미만으로너무적은경우가있 는데, trisomy 13, 18, triploidy, monosomy X 의경우작은 태반으로인해태반으로부터유래한 cfdna 가적을수있다 [33-35]. 따라서이럴경우 NIPT 를반복하는것말고도자 세한정밀초음파와함께침습적인검사를받을수있는기회 도주어야한다. 4. Other chromosome abnormalities 기존의선별검사를통해침습적인검사를했을경우발견 되는 16.9-23.4% 의염색체이상은 NIPT 로는선별이되지 않는다 [36,37]. 따라서심장기형등의주요기형이초음파로 발견된경우라면우선적으로융모막검사나양수검사등의 침습적인검사를고려하는것이좋다. 5. Multiple pregnancy NIPT 를쌍태임신에서적용한여러연구들이있었고, 기 존의모체혈액이나초음파를이용한선별검사보다는우수 한결과를보여주었다 [38-41]. 그렇지만아직단태아만큼 충분한증거가없기때문에 NIPT 는단태임신에서우선적으 로고려되고쌍태임신을포함한다태임신에서는더많은연 구가필요하다 [42]. Clinical Application of Non-invasive Prenatal Test NIPT 를실제임상에어떻게적용할것인가에관한많은논 란이있다. 검사를제공하는회사들에서는모든산모들에게 검사를제공하기를원하고, 대부분의전문가단체에서는고위 험군 ( 분만시산모의나이가 35 세이상, 기존의선별검사에서 고위험군으로나온경우, 부모의염색체이상으로다운증후군 이나파타우증후군고위험군인경우, 이전에다운증후군같은 염색체이상이있었던경우등 ) 에서만 NIPT 를고려할것을권 고한다 [43]. 현재시행되고있는여러가지검사들중에어떤 것이가장뛰어난검사인지에관한연구도아직없다. 998 대한의사협회지
Kim K Maternal serum cell free DNA test for aneuploidy Table 2. Positive predictive value of non-invasive prenatal test according to maternal age [42] 1. Strategies [44] 1) Primary non-invasive prenatal test 산모의위험인자와관계없이모든산모를대상으로 NIPT 를시행하는전략이다. 가장많은다운증후군을산전에발견 할수있지만고비용등의문제로인해 ISPD (International Society for Prenatal Diagnosis) 를제외한대부분의가이드 라인에서는, 이방법을지지하지않는다 [42,45]. 2) Secondary non-invasive prenatal test 기존의혈액검사와초음파검사로고위험군으로선별된사 람들에게 2 차적선별검사로 NIPT 를적용하는모델이다. 다 운증후군발견율은 primary NIPT 에비해떨어지지만 90% 이상의침습적인검사를줄일수있고, 전체적인비용면에 서도유리하다. 3) Contingent non-invasive prenatal test Primary 와 secondary 의중간단계의모델로기존의선별 검사에서고위험군과, 중간위험군에 NIPT 를적용하는모델 이다. Primary NIPT 에비해다운증후군의발견율도비슷하 게유지하면서비용을줄일수있는장점이있다. 국가적인 산전진단프로그램을운영하고있는영국등에서는가장관 심을가지는모델이다 [46]. Age 25 years Age 40 years Trisomy 21 33 87 Trisomy 18 13 68 Trisomy 13 9 57 Values are presented as %. 1) Pre-test counseling NIPT는다운증후군, 에드워드증후군, 파타우증후군, 터너증후군같은흔한염색체이상에대한선별검사로기존의선별검사보다정확한검사이지만위양성과위음성의가능성이있음을설명해야한다. 기존의선별검사보다고비용이들고, 산전에발견되는염색체이상의 16.9-23.4% 는 NIPT로발견할수없다 [36,37]. NIPT는신경관결손증에대한 AFP 검사와 nuchal translucency를포함한초음파검사를대신할수는없다. NIPT의장점, 단점, 한계등을설명하고환자로부터 informed consent를받아야한다. 2) Post-test counseling 고위험군으로나온경우위양성의가능성이있으므로반드시융모막검사나양수검사로확인을해야한다. NIPT 결과만으로돌이킬수없는산과적인결정을하면안된다. NIPT의양성예측도는 Table 2와같다 [42]. 예를들어 25세의여성이 NIPT 검사상다운증후군고위험군으로나왔을경우태아가진짜다운증후군일가능성은 33% 이고 67% 는정상이라는의미다. 나이와임신주수, 검사방법에따른검사결과의양성예측도를간단히인터넷으로알려주는사이트도있다 (http://mombaby.org/nips_calculator.html) [52]. 저위험군으로나온경우에도신경관결손증검사와초음파검사를포함한기존의산전검진은여전히중요하고, 초음파검사에서심장기형과같은이상소견이발견된경우에는 NIPT가저위험군이라고하더라도양수검사와같은침습적인검사의대상이된다. 2. Pre-test and post-test counseling 2011년중국과미국에서이윤을추구하는기업의주도로 NIPT가상업적으로제공됨에따라여러전문가단체에서 NIPT의임상적사용에대한가이드라인을발표하였다 [42,43,47-51]. 거의대부분의가이드라인에서현재 NIPT 는모든산모를대상으로실행하지말고염색체이상의위험도가높은군에서고려할것을권고하고있고, 무엇보다 NIPT를환자에게제공할때는검사전후에전문가에의한상세한상담을권고하였다. 고찰 NIPT는 trisomy 13, 18, 21을임신 10주라는이른시기부터비교적정확히선별해내는좋은검사임에는틀림이없다. 현재국내에서는아직제도적인장치가완전히마련되지않았고, 산부인과학회나유전의학회등의전문가단체에의한가이드라인도없는실정이다. 이윤을추구하는기업에의해서 NIPT가주도되고, 특허와지적재산권에대한신청이늘어나면서검사의가격이올라가고일반인의 모체혈장내유리 DNA 를이용한비침습적산전검사 999
J Korean Med Assoc 2015 November; 58(11): 995-1002 접근성이떨어질수도있다. 현재환자에게직접검사가시도되진않지만, 광고를통한광범위한마케팅으로충분한사전지식없는환자에게검사가행해질수있다. 적절한규제없이시장경제논리에만맡겨질경우검사의질에심각한문제를초래할수도있다. 또한윤리적문제로는 NIPT 로인해결국낙태되는염색체이상아이가증가할수도있고, 치료하는것에는소홀하게될수있다. 가장큰문제중의하나로 NIPT는분명한선별검사임에도불구하고 NIPT 검사결과가고위험군으로나온경우 6.2% 에서는융모막검사나양수검사같은확진검사없이임신을종결시킨것으로보고되었다는것이다 [34]. 분자유전학기술의발전으로태아에대한너무많은정보를출산전에알수있게됨에따라부모를불필요하게불안하게할수도있다. NIPT처럼안전하고, 쉬운검사일수록 informed consent를통한자발적인선택이잘이루어지지않을수도있다 [53]. 예상되는많은문제점들을해결하기위해서는전문가단체와보건당국에의해우리나라에가장적합한가이드라인을만들어야하고, 검사를제공하는의료인에대한교육도시급한과제이다. 결론 NIPT는기존의다운증후군선별검사보다낮은위양성율로높은발견율을보이는훌륭한검사임에는분명하다. 그렇지만전술한바와같이여러가지한계점을가지고있고, 고비용이드는검사이므로현재로서는고위험군산모들을대상으로우선적으로시행하고, 검사전후에담당선생님에의한적절한유전상담이매우중요하다. 또한 NIPT 검사결과가고위험군으로나온경우, 이결과만으로돌이킬수없는산과적인결정을하지말고, 반드시융모막검사나양수검사로확인검사를해야한다. 찾아보기말 : 다운증후군 ; 모체혈청선별검사 ; 세포유리 DNA; 비침습적산전검사 ORCID Kunwoo Kim, http://orcid.org/0000-0002-2272-5665 REFERENCES 1. Weijerman ME, de Winter JP. Clinical practice: the care of children with Down syndrome. Eur J Pediatr 2010;169:1445-1452. 2. Nicolaides KH. Screening for fetal aneuploidies at 11 to 13 weeks. Prenat Diagn 2011;31:7-15. 3. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485-487. 4. Chan KC, Zhang J, Hui AB, Wong N, Lau TK, Leung TN, Lo KW, Huang DW, Lo YM. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88-92. 5. Lun FM, Chiu RW, Chan KC, Leung TY, Lau TK, Lo YM. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem 2008;54: 1664-1672. 6. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing. Clin Chem 2010;56:1279-1286. 7. Nygren AO, Dean J, Jensen TJ, Kruse S, Kwong W, van den Boom D, Ehrich M. Quantification of fetal DNA by use of methylation-based DNA discrimination. Clin Chem 2010;56: 1627-1635. 8. Sikora A, Zimmermann BG, Rusterholz C, Birri D, Kolla V, Lapaire O, Hoesli I, Kiefer V, Jackson L, Hahn S. Detection of increased amounts of cell-free fetal DNA with short PCR amplicons. Clin Chem 2010;56:136-138. 9. Nicolaides KH, Syngelaki A, Ashoor G, Birdir C, Touzet G. Noninvasive prenatal testing for fetal trisomies in a routinely screened first-trimester population. Am J Obstet Gynecol 2012;207:374.e1-6. 10. Ashoor G, Poon L, Syngelaki A, Mosimann B, Nicolaides KH. Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks' gestation: effect of maternal and fetal factors. Fetal Diagn Ther 2012;31:237-243. 11. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999;64:218-224. 12. Kitzman JO, Snyder MW, Ventura M, Lewis AP, Qiu R, Simmons LE, Gammill HS, Rubens CE, Santillan DA, Murray JC, Tabor HK, Bamshad MJ, Eichler EE, Shendure J. Noninvasive whole-genome sequencing of a human fetus. Sci Transl Med 2012;4:137ra76. 13. Fan HC, Gu W, Wang J, Blumenfeld YJ, El-Sayed YY, Quake SR. Non-invasive prenatal measurement of the fetal genome. Nature 2012;487:320-324. 14. Lun FM, Chiu RW, Sun K, Leung TY, Jiang P, Chan KC, Sun H, Lo YM. Noninvasive prenatal methylomic analysis by genomewide bisulfite sequencing of maternal plasma DNA. Clin Chem 2013;59:1583-1594. 1000 대한의사협회지
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