The utility of molecular techniques for better prenatal diagnosis services in Romania

Romanian Biotechnological Letters Vol.17, No.4, 2012 Copyright © 2012 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 17, No. 4, 2012 7515

The utility of molecular techniques for better prenatal diagnosis services in Romania

Received for publication, May 18, 2011

Accepted, January 30, 2012 ADRIANA STAN1, CRISTINA DRAGOMIR1, DANIELA TUDOR1, LORAND SAVU1, EMILIA SEVERIN2* 1Genetic Lab, Bucharest, Romania 2Carol Davila University of Medicine and Pharmacy, Bucharest, Romania *[email protected]

Abstract

Objectives: Our aims were to evaluate the current status of prenatal diagnosis in Romania and based on our five years of practice and data to offer the best practice protocol for health-planners and policy decision-makers in order to improve the quality of prenatal care, screening and diagnosis services.

Design: 2740 samples including amniotic fluids, CVS, products of conception and blood from female patients with high risk pregnancy were tested. Samples were tested using QF-PCR for rapid diagnosis of trisomy 13, 18, 21 and sex chromosome aneuploidies followed by G-banding chromosome karyotype analysis from cultured cells. Results: We detected 190 (6.93%) abnormal results from which the majority were autosomal anomalies (n=148), 22 cases (0.80%) were sex chromosomes aneuploidies and in 20 cases (0.72%) all five chromosomes were involved. 42.1% of the autosomal abnormalities were represented by trisomy 21 cases (n=80). Normal results were seen in 2550 (93.5%) cases. No false positives or false negative results were detected.

Conclusions: We consider this rapid method to be a reliable and accurate tool in relieving maternal anxiety providing a diagnosis report in maximum 24 hours after sampling. QF-PCR assay was also efficient in detecting maternal cell contamination of fetal sample, submicroscopic duplication, somatic microsatelite mutation and mosaicism. Analyzing the difference in methodology and results we offer an efficient strategy for the health prenatal care based on medical, social and economic benefits for both population and parents.

Key words: rapid aneuploidy diagnosis (RAD), fetal chromosome pathology, prenatal health care. Introduction

Romania is a European country with an upper-middle income economy, low birth rate and high rate of young population migration abroad [1]. With an aging population and a tendency to delay the birth of a first child, Romania should adopt new strategies for increasing the birth rate but also to prevent pregnancy complications caused by advanced maternal age.

It is well-documented that a woman’s risk of having a baby with certain birth defect involving chromosome increases with age. In this respect, the reproductive medicine and the prenatal care are important objectives for national health policy. The current policy for prenatal screening and diagnosis in Romania includes: a national programme for all pregnant women over age 35 at conception offering tests free of charge; biochemical screening, ultrasound and CVS/amniocentesis are performed in 6 public medical centers throughout the country. Prenatal screening / diagnosis is also offered to all pregnant women independently of maternal age; costs are covered by national health insurance. Many public laboratories perform conventional cytogenetics but rapid aneuploidy test are offered in few private labs. So, the aims of the study were to evaluate the current status of prenatal diagnosis in Romania and based on our five years of practice and data to offer the best rapid protocol for health-planners and policy makers in order to improve prenatal care, screening and diagnosis services.

ADRIANA STAN, CRISTINA DRAGOMIR, DANIELA TUDOR, LORAND SAVU, EMILIA SEVERIN

7516 Romanian Biotechnological Letters, Vol. 17, No. 4, 2012

The high frequency of occurrence of genetic abnormalities in human population made prenatal diagnosis the only prevention strategy but also a cause of increasing rate of abortion. Pregnant women are informed in time by the obstetricians about the existence of all the prenatal tests available, mentioning the advantages, the related risks and possible difficult decisions arising from these tests. When an aneuploidy is discovered after prenatal diagnosis, the primary reaction of the parents is often to terminate the pregnancy. High risk pregnant women are not encouraged to terminate the pregnancy because of fetal abnormality. Genetic counseling and support services are offered where the reproductive implications and prenatal diagnosis can be discussed.

Modern techniques, such as FISH, QF-PCR or MLPA, applied in the prenatal diagnosis field allow us to obtain valid informations about fetus’s genetic status from the early days of development. The quantitative fluorescence PCR (QF-PCR) assay has been used for more than one decade in the prenatal diagnosis field, allowing the rapid detection of selected chromosomal aneuploidies. Specific studies revealed that the main category of chromosomal abnormalities prenatally detected is aneuploidy involving autosomes and sex chromosomes [2-4]. The most common autosomal anomaly is trisomy 21 or Down syndrome, a well known genetic disorder causing heart diseases, developmental delay and learning disabilities. Trisomy 21 has an incidence estimated at 1:600-800 births, affecting more than 30000 people in Romania [5]. Trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) are the next more frequently autosomal abnormalities detected in prenatal testing; both conditions leads to early neonatal death caused by multiple congenital abnormalities [2]. Sex chromosomes aneuploidies have a high incidence of occurrence affecting 1 in 400-500 live newborns mostly because of the relative less severe condition [6]. Some sex chromosome abnormalities can have mild-moderate mental retardation/learning disabilities as a feature and affect mainly the development of sexual characteristic. The prenatal diagnosis panel recommends the maternal serum screening tests (MSS) and ultrasonography for the first and the second trimester of pregnancy. These prenatal screening methods help to select the high risk pregnancies for chromosomal abnormalities and neural tube defects [7]. Following these blood tests, women with high risk pregnancy undergo invasive sampling procedures such us amniocentesis, chorionic villus or fetal blood sampling for prenatal diagnosis. The invasive prenatal procedures are routinely performed for prenatal diagnosis by the standard cytogenetic technique - karyotype of in vitro cultured fetal cells [8-10]. The karyotype detects numerical and structural chromosomal abnormalities; regarding the structural genetic anomalies, fetal karyotype detects it only when involves more than 4 Mb so that small fragments can be missed [11]. This procedure has also special requirements including in vitro fetal cells cultivation about 13-14 days that causes an inevitable anxiety period for the pregnant woman, high quantity of amniotic fluid (16-20 ml) and also high laboratory quality control because of the increased risk of culture bacterial or fungal infections [12-14]. These requirements become the major disadvantages especially the time delay between sampling and diagnosis. The quantitative fluorescent polymerase chain reaction (QF-PCR) method was introduced in the prenatal diagnosis field to overcome the need to culture fetal cells for rapid diagnosis of trisomy 13, 18, 21 and sex chromosome aneuploidies [11,13-14]. QF-PCR assay has been used extensively by several research teams in the last years and proved to be the best alternative method for prenatal diagnosis of common chromosome aneuploidies because the results are reliable and also ready in a few hours after sampling, using small amounts of fetal sample [9,11,13,14]. The QF-PCR technique involves the



relative quantification of microsatellite alleles to determine sequence copy number, using fluorescence labeled primers [16]. Materials and Methods We tested 2740 prenatal samples from female patients with high risk pregnancy (advance maternal age, previous child with aneuploidy, known parental rearrangement, suspicious ultrasound findings or biochemical screen risk) that had undergone invasive sampling procedures for prenatal diagnosis and also fetal tissue from miscarriages. All samples were collected between December 2007 and August 2011. We received mostly amniotic fluid samples (92.4%) collected between 13 and 23 weeks of gestation but also fetal tissue from chorionic villi samples (3.36%), miscarriages (3.18%) and blood (1.06%). For blood-stained amniocytes pellets and fetal tissue samples we first proceed with a wash step to remove/reduce maternal blood contamination. Fetal genomic DNA extraction was performed from 1-2 ml of amniotic fluid, small fetal tissue samples of 2-5 mm or from 5-10µl fetal blood. We performed the DNA extraction with the DNA IQ™ System kit and the QIAamp® DNA Mini kit, according to the manufacturers’ recommendations (Promega and respectively Qiagen). The DNA elution was performed in 25-50 µl from which approximately 3-30 ng DNA were used in a QF-PCR multiplex based reaction. We used three different products for diagnosis of chromosome 13, 18, 21, X and Y aneuploidies: the Aneufast™ kit (Genomed Ltd, UK), the Devyser Compact (DevyserAB) and an “in house” product developed based on the published data [17]. All three products were based on the quantitative fluorescent amplification principle of selected chromosome-specific short tandem repeats (STRs) and non-polymorphic markers. We listed and compare in the table 1 and 2 the STR markers included in the three QF-PCR tests.

Table 1. STR markers for chromosome 13, 18 and 21 included in the Aneufast™ kit (Genomed Ltd, UK), the Devyser Compact (DevyserAB) and Mann et al.method.

Chromosome Location Aneufast Devyser Compact

Mann et al. method

13 13q12.1 § 13q12.12 § § § 13q12.1-14.1

§

13q13.3 § 13q14.3 § 13q21 § 13q21.32 § 13q21.33 § 13q31.1 § 13q31.3 § § 13q31-32 § §

18 18pter-11.22

§

18p11.31 § § 18q11.2 § § 18q12.2 § 18q12.3 § § § § 18q21.1 §



18q21.31 § 18q21.32-33

§

18q22.1 § § § 18q22.2 § 18q22.3 §

21 21q21 § § 21q21.1 § § § § § 21q21.2 § 21q21.3 § § § 21q22.1 § 21q22.13 § 21q22.2 § 21q22.3 § § § 21q22.3-ter

§

Table 2. STR markers for the sex chromosomes included in the Aneufast™ kit (Genomed Ltd, UK), the Devyser Compact (DevyserAB) and Mann et al. method.

Chromosome Markers location

Aneufast Devyser Compact

Mann et al.

method X and Y chr. Non-polimorphic markers

Xp22.22/Yp11.2 § § Xp22.1-22.31/Yp11.2

§

Pseudoautosomal markers

Xq21.3/Yp11.31 § Xq21.31/Yp11.31 § Xp22.32/Yp11.3 § Xp28/Yq §

X Xp22.3 § § Xq § Xq11.2-12 § Xq12-12.33 § Xq13.1 § § Xq21.33 § Xq26.1 § Xq26.2 § § § Xq28 § §

Y Yp11.2 § Yp11.31 § § Yq11.223 §

X/7 Xq13/7q34 §

The QF-PCR technique involves the usage of fluorescently labeled primers to determine the specific copy number of each marker based on the assumption that the amplified sample is directly proportional to the target sequence amount in the sample. The PCR products ware separated and visualized by capillary electrophoresis using the automated ABI PRISM® 310 Genetic Analyzer (Applied Biosystems). The raw data were interpreted by the GeneMapper® ID Software v3.2, Applied Biosystems. The results interpretation was performed based on the criteria presented in the “Profesional guidelines for clinical cytogenetics and clinical molecular genetics - QF-PCR for the diagnosis of aneuplidy best practice guidelines” [18].



Results We used the Aneufast™ kit (Genomed Ltd, UK) [19] for 1257 samples and then we changed with the Devyser Compact (DevyserAB) [20] for the next 406 samples. Since June 2010 we analyzed 1077 samples using a method previously described by Mann et al. in 2008. The first 200 prenatal samples included in this study were collected from December 2007 to April 2008 and were considered the QF-PCR method validation test samples. We analyzed it by both routinely cytogenetic techniques (fetal karyotype and FISH) and QF-PCR approach. The assay was performed without knowing the karyotype and FISH results; we obtained the confirmation in all 200 tests included in this laboratory validation procedure. Ninety three and one-half percent of the 2740 samples tested were normal with at least two informative (heterozygous) markers on every chromosome tested; three samples that showed just a single informative marker for one chromosome were reported as normal and the results were cytogenetically confirmed; two sample were uninformative for all markers of one chromosome and the final report was released by the karyotype. In three chorionic villi samples we detected only the maternal DNA profile. We detected 190 (6.93%) abnormal results from which the majority were autosomal anomalies (n=149), 22 were sex chromosomes aneuploidies and in 20 cases all five chromosomes were involved. 42.1% of the autosomal abnormalities were represented by trisomy 21 cases (n=80). We also detected trisomy 18 (n=23), trisomy 13 (n=7) and sex chromosomes aneuploidies including 47, XXY (n=3) and 47, XXX (n=2). Regarding the monosomy X cases (n=7) we used for confirmation the Devyser Compact kit that includes the discriminating marker 7X (7q34/Xq13). Recently we included the paralogous sequence TAF9L (Xq21.1/3p24.2) previously reported by Deutsch et al. [21] for monosomy X detection in cases were all X chromosome specific markers were noninformative. We reported 17 fetuses with triploidy and observed also submicroscopic duplication, somatic microsatellite mutations and mosaicism cases. The maternal cells contamination (MCC) was observed in one third of al samples. The majority of fetal samples presenting MCC were not visibly bloodstained; in these cases the MCC was detected in the amniocytes pellets after centrifugation step and we proceed with a wash step. In 4.75% of all cases the QF-PCR analysis showed different levels of maternal DNA contamination (second genotype) appearing as extra allele peaks or/and inconsistent dosage ratios for each chromosomes [22, 23]. The result was issued after the comparison between the fetal/maternal genetic profile and the maternal DNA profile. Some amniotic fluids cell sediment presented a brown/green color as a sign of an old bleeding event [16]. In all MCC samples it was possible to detect fetal sex regardless of MCC level, but in 20 cases the MCC level exceeded 50% and no result was reported but fetal genetic sex. In MCC cases the fetal karyotype reported a result consistent with the selection and growth of fetal cells and loss of maternal cells during the culture process [16], except two CVS where it was a disagreement with the QF-PCR result. In both cases the QF-PCR reported normal results with maternal DNA contamination less the 50% and male genetic gender. The karyotype showed normal results and female gender fetuses. We conclude that in the process of fetal tissue culturing the maternal cell line completely overgrew the culture, thereby leading to diagnostic errors, including incorrect sex determination [24]. Submicroscopic polymorphic duplications of microsatellites (submicroscopic imbalance) were observed in 14 cases appearing as a single marker consistent with trisomy - trisomic triallelic (Figure 1) or diallelic patterns - while all other informative markers were normal [22, 25-26]. SMD profiles were detected in D13S631, D13S252, D13S634, D18S386, DXYS218, X22.



Figure 1.The triallelic D13S631 marker in mother and in fetus. To distinguish between a benign submicroscopic polymorphic duplication and a partial trisomy due to unbalanced translocation we proceed to analyze the parental blood samples. In 8 cases we established the pattern of inheritance of the CNV (copy number variant) and therefore unlikely to be of clinical significance [20, 22]. In 6 cases we conclude that the single trisomic marker appeared “de novo”. The karyotype was available for 7 samples and reported normal results. The CMGS/ACC Best Practice Guidelines v2.01 also mention that abnormal markers that are flanked by normal markers may represent CNV. In two cases with fetal malformations detected by ultrasound scanning no other test then QF-PCR was requested and after reporting the single trisomic “de novo” D13S631 marker, the pregnancies were terminated. In one case with a SMD result on chromosome 18, the karyotype confirmed a chromosome 18 mosaicism.

Figure 2.The tetranucleotid D21S1435 marker exibitiong a SMM.

In 27 cases we detected a single marker with a triallelic pattern of unequal peak areas previously reported as somatic microsatellite mutation (SMM) [25]. The mutated marker showed the areas of the two lowest alleles combine to equal the higher allele [25] Figure 2. All other informative markers gave normal diallelic results. The STR markers that presented such pattern were D21S1411, D21S1435, D21S1446, D18S386, D13S634, D13S305, D13S628 and D13S631. For eleven cases the karyotype was available and the results were normal. For three cases we establish the inheritance pathway.

We detected one amniotic fluid sample the D21S1446 marker with an abnormal allelic ratio of approximately 3:1 (Figure 3). We analyzed both parents and the mother presented the



same abnormal D21S1446 pattern. We conclude that this a SMM pattern and the 221bp allele in the fetus genetic profile was inherited from the mother. The conventional karyotype report indicated a normal fetus.One case involving the D21S1435 marker with a skewed diallelic result was considered a SMM. The fetal karyotype result was normal (Figure 4).

Figure 3.Case report - the diallelic D21S1446 marker in mother, father and in fetus.

Figure 4.Case report - the diallelic D21S1435 marker in mother, father and in the fetus. The primer site polymorphism resulting in the complete or partial allelic dropout was

excluded in all SMD and SMM cases by repeating the PCR with a lower annealing temperature [25].

We detected 10 mosaicism cases: three for trisomy 21, five for sex chromosomes and two cases involving all five chromosomes tested. One of the chromosome 21 mosicism cases was a confined placental mosaicism confirmed by the normal result of the fetal blood karyotype. Regarding the sex chromosomes mosaicism we were able to detect five cases: three were confirmed by karyotype - 45,X/47,XXX, 47,XXX/46,XX and 45,X/47,XYY and for the other two cases no test but QF-PCR was available. Two products of conception



presented a triploidy mosaicism; the karyotype was requested for both sample but, the result was available for just one of them and confirmed the QF-PCR report. For a chromosome 18 mosaicism the QF-PCR detected a single diallelic marker consistent with duplication and the karyotype result confirmed the mosaic status of chromosome 18. Being a CVS sample we will proceed with the fetal karyotype from amniotic fluid to eliminate the possibility of confined placental mosaicism [27]. Regarding the QF-PCR test incapability to detect some mosaicism when the extra cells line represent less than 15% in cases of triallelic trisomy and 20% in diallelic trisomy [28] we mention two cases. For a patient exhibiting pseudohermaphroditism the QF-PCR result was normal, instead the karyotype report was 45,X/46,X,t(5p2;Yq). The second case was an amniotic fluid sample were the QF-PCR detected the absence of the DYS488 marker (Yq11.223) and the karyotype report was 45,X/46,XY,delY. 87 products of conception were analyzed by our laboratory. 71 samples showed normal results and 15 were abnormal: triploidy (n=5), trisomy 18 (n=4) and one case of partial trisomy 18, trisomy 21 (n=1), trisomy 13 (n=1), monosomy X (n=1), complex mosaicism involving all five chromosomes (n=2). One sample showed only the maternal profile and the result was not reported. We were not able to give a result for one sample because it was formalin-fixed and the PCR reaction was inhibited by the chemical modifications that occur in DNA and in protein (DNA-protein cross-linkage) [29]. When requested, the karyotyping procedure failed to give a result in 5 cases because of the cultures infection. Considering that the main cause of spontaneous miscarriages is represented by genetic abnormalities our normal findings in most cases suggest that further testing should be made [30]. Uniparental disomy (UPD) was not excluded because both parents were not tested. From all 2740 samples tested by QF-PCR, 1344 were also analyzed by karyotype. 42 samples with normal QF-PCR result were found with other chromosomal abnormalities following karyotype analysis (Table 3). We evaluate the test sensitivity and for the 1344 sample tested by both classical and rapid tests it was 81%. The test showed 100% specificity with a positive predicted value of 100% and a negative predictive value of 96.5%. Since we started performing the rapid prenatal detection of chromosomal abnormalities by QF-PCR, the number of requests outgrew our expectation. Considering that we started at the end of 2007 we will count starting with 2008. As is showed in the graph bellow (Table 4), QF-PCR is at the moment the prenatal test preferred by most pregnant women and physicians thanks to is advantages offered. In the year 2008 the QF-PCR tests were mostly requested along with the karyotype. This changed over the next two years and the QF-PCR tests exceeded the number of all other prenatal tests requested in our laboratory. The FISH request decreased constantly in this period of time and the karyotypes are currently performed along with QF-PCR to reduce maternal distress in the waiting process for the release of the karyotype result. Discussion The QF-PCR based assay for the rapid prenatal diagnosis of trisomy 21, 18, 13 and sex chromosome aneuploidies proved to be a test of great value. The 2740 sample tested in Romania by our laboratory showed that the QF-PCR method is rapid and accurate, considering it as an alternative to the conventional karyotype analysis. The test had a very good feedback from anxious pregnant women with positive serum screening results that were receiving a QF-PCR confirmation in approximately 24 hours after sampling. From the 2740 sample tested by QF-PCR assay, 1344 were analyzed also by karyotype and there were no false positive or false negative results. In our laboratory for 1187



samples our partners requested only the QF-PCR analysis as prenatal test and when the classical approach encountered difficulties in the amniocytes harvesting, often, the QF-PCR remained the only alternative. An important advantage was the possibility of a correct diagnosis in case of MCC when the maternal cells do not exceeded 50% and in the majority of the mosaicism cases. Based on the conclusions presented in this paper we are confident that the QF-PCR test can reduce the number of karyotype in pregnancies carefully evaluated by ultrasound and maternal serum screening tests. In our experience the QF-PCR assay is of great value considering the rapid release of the result, the 100% accuracy in detecting non-mosaic aneuploidies of chromosomes 13, 18, 21, X and Y and also the financial aspect considering that the QF-PCR is less then half-priced of the karyotype. In Romania another important aspect is the transportation conditions because we receive samples all over the country. The lack of specific requirements of temperature and time till the sample is received at the laboratory is a major advantage for QF-PCR test . Conclusions At the moment the QF-PCR as a stand-alone prenatal test is preferred in carefully monitored pregnancies and we expect this year that the QF-PCR request to exceed the all others prenatal tests put together. We recognize that this approach has its limitations (aneuploidies outside of the test panel) but its advantages make the QF-PCR test the preferential choice in prenatal diagnosis in Romania. Analyzing the difference in methodology and results we offer an efficient strategy for the health prenatal care based on medical, social and economic benefits for both population and parents. Acknowledgements The authors thank all families for their voluntary participation in this study and Dr. Dinu Albu for providing us amniotic fluid samples. References 1. ROMANIA IN FIGURES 2011, Population, 10

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The utility of molecular techniques for better prenatal diagnosis services in Romania

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