Clinica Chimica Acta 445 (2015) 2–6

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Detection of sex chromosome aneuploidies using quantitative fluorescent PCR in the Hungarian population Balint Nagy ⁎, Richard Gyula Nagy, Levente Lazar, Julianna Schonleber, Csaba Papp, Janos Rigo Jr. 1st Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary

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Article history: Received 2 October 2014 Received in revised form 9 March 2015 Accepted 9 March 2015 Available online 16 March 2015 Keywords: Quantitative fluorescent-PCR Sex chromosomes Small tandem repeats Aneuploidies Prenatal diagnosis

a b s t r a c t Background: Aneuploidies are the most frequent chromosomal abnormalities at birth. Autosomal aneuploidies cause serious malformations like trisomy 21, trisomy 18 and trisomy 13. However sex chromosome aneuploidies are causing less severe syndromes. For the detection of these aneuploidies, the “gold standard” method is the cytogenetic analysis of fetal cells, karyograms show all numerical and structural abnormalities, but it takes 2–4 weeks to get the reports. Molecular biological methods were developed to overcome the long culture time, thus, FISH and quantitative fluorescent PCR were introduced. In this work we show our experience with a commercial kit for the detection of sex chromosome aneuploidies. Methods: We analyzed 20.173 amniotic fluid samples for the period of 2006–2013 in our department. A conventional cytogenetic analysis was performed on the samples. We checked the reliability of quantitative fluorescent PCR and DNA fragment analysis on those samples where sex chromosomal aneuploidy was diagnosed. Results: From the 20.173 amniotic fluid samples we found 50 samples with sex chromosome aneuploidy. There were 19 samples showing 46, XO, 17 samples with 46, XXY, 9 samples with 47, XXX and 5 samples with 47, XYY karyotypes. The applied quantitative fluorescent PCR and DNA fragment analyses method are suitable to detect all abnormal sex chromosome aneuploidies. Conclusions: Quantitative fluorescent PCR is a fast and reliable method for detection of sex chromosome aneuploidies. © 2015 Published by Elsevier B.V.

1. Background Conventional karyotyping is the “gold standard” method for the prenatal detection of numerical and structural chromosomal abnormalities in diagnostic centers. The culture of the amniotic fluid cells takes time and the reporting lasts about 2–3 weeks. Alternative methods have been developed to solve this problem. Fluorescence in situ hybridization (FISH) was developed in 1970s, and the first centromerspecific probes were used for the detection of the most common trisomies from 1990 [1–3]. FISH probes are expensive and the method is labor intensive, whereas faster and cheaper methods such as quantitative fluorescent PCR and DNS fragment analysis (QF-PCR) were introduced in 1993 [4]. These two methods give information according to the designed probes or primer systems but not for the whole chromosomal set. Array comparative genomic hybridization (aCGH) method was introduced about ten years ago which gives information about all chromosomes with high resolution, which is better from the conventional karyotyping [5]. In the past few years the next generation sequencing and the use of ⁎ Corresponding author at: 1088 Budapest, Baross u. 27, Hungary. E-mail address: [email protected] (B. Nagy).

http://dx.doi.org/10.1016/j.cca.2015.03.009 0009-8981/© 2015 Published by Elsevier B.V.

massive parallel sequencing made possible the non-invasive detection of the most common trisomies. Non-invasive prenatal testing using fetal DNA from maternal plasma became very popular lately. Several tests showed that these are highly accurate for fetal trisomy evaluation in high risk pregnancies [6–9]. Nowadays the most commonly used tests in the routine prenatal diagnostic testing centers are karyotyping, QF-PCR and FISH. We started the prenatal genetic diagnosis of chromosomal abnormalities in our laboratory with karyotyping in 1991 and QF-PCR was introduced in 1997 [10,11]. This method makes reliable detection of the most common autosomal and sex chromosome trisomies, while the detection of Turner syndrome, and XXX syndrome was problematic. Recently new commercial tests were developed to solve this problem. We decided to check out the accuracy of one of these commercially available tests on our samples collected in the last 8 years.

2. Materials and methods 21.173 amniotic fluid samples were collected at the 16–20th gestational weeks during the last 8 years (from January 1st 2006, until

B. Nagy et al. / Clinica Chimica Acta 445 (2015) 2–6 Table 1 The diagnosed numerical chromosomal aneuploidies from 2006 to 2013. Year

2006 2007 2008 2009 2010 2011 2012 2013

Number of amniocentesis

2554 2981 3146 2858 2279 2604 2047 1704 20.173

Autosomal numerical aneuploidy

X and Y chromosome numerical aneuploidy

Trisomy 21

Trisomy 18

Trisomy 13

XO

XXY

XXX

XYY

22 24 22 28 20 31 21 24 192

6 7 7 9 8 4 7 12 60

4 1 2 3 2 5 2 3 22

2 3 4 3 4 1 1 1 19

1 3 3 2 0 2 6 0 17

0 2 1 1 1 1 0 3 9

0 0 0 0 1 1 2 1 5

December 31st 2013) in our department (Table 1). All patients were informed and they signed a consent. 2.1. Karyotyping Amniotic fluid centrifugation and culture were performed immediately after sample was obtained. Briefly, 10 ml of amniotic fluid samples were centrifuged at 1200 rpm for 10 min to obtain amniocytes, and the supernatant was discarded. Cells were cultured in Chang Medium D (Irvine Scientific, Ireland) for 10–14 days [12]. Conventional cytogenetic analysis was performed on all samples by using Lucia automatic evaluation system (Prague, Czech Republic). 2.2. DNA isolation DNA was isolated from 1.5 ml amniotic fluid samples by using High Pure PCR Template Preparation kit (Roche, Penzberg, Germany) according to the manufacturer's instructions [13]. 2.3. Quantitative fluorescent PCR and DNA fragment analyses (QF-PCR) QF-PCR was performed on those selected samples where we detected sex chromosome related numerical abnormality. We used the

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Chromoquant QF-PCR STaR kit according to the manufacturer's instructions (CyberGene AB, Stockholm, Sweden). This kit contains small tandem repeat (STR) sequences for five chromosomes, 4 STRs for chromosome X, 3 STRs for chromosome Y, 6 STRs for chromosome 21, 5 STRs for chromosome 18 and 5 STRs for chromosome 13. PCR was performed on an Applied Biosystems 9800 (Perkin Elmer, USA) thermal cycler. The obtained PCR products were analyzed on an Applied Biosystems Genetic Analyzer 3130 (ABI, USA) with a molecular weight standard ABI LIZ 500 (ABI, USA). The analyzed fragments were divided to normal euploid or triploid according to the peak ratios (euploid N 0.8 b 1.4, while triploid N1.8; or b 0.65). 3. Results During the past 8 years we performed 20.173 amniocenteses in our department and we found 274 fetuses with autosomal and 50 fetuses with sex chromosome aneuploidies (Table 1). We checked out the reliability using a commercially available QF-PCR kit for detection of sex chromosome aneuploidies on these samples. We found 19 samples with 45, XO, 17 samples with 47, XXY, 9 samples with 47, XXX and 5 samples with 47, XYY karyotypes (Table 1). Fig. 1 shows a typical electrophoretogram of a sample with 45, XO karyotype. The TAF9B peaks (3p24.2/Xq13.1–q21.1) are showing 1:0.5 ratio. Fig. 2 is an electrophoretogram of a sample having a 47, XXY karyotype. The AMEL gene peaks are showing 1:0.5 ratio. Fig. 3 shows a sample with 47, XYY karyotype. The AMEL gene peaks are present in 1:2 ratio. Fig. 4 is a typical electrophoretogram of a sample having a 47, XXX karyotype. All X chromosome related marker shows 1:1:1 or 1:0.5 or 0.5:1 ratio. The QF-PCR kit is able to detect all observed sex chromosomal aneuploidies. Based on our results the occurrence of 45, XO is 0.0009; 47, XXY is 0.0008; 47, XXX is 0.0004 and 47, XYY is 0.00002 in the Hungarian population. 4. Discussion The most common numerical chromosomal abnormalities observed in the liveborns are trisomy 21, 18, 13, and sex chromosomes [14–16]. The most widely used method for prenatal detection for these is the cytogenetic analyses of fetal cells obtained by amniocentesis or

Fig. 1. Electrophoretogram of a fetus with 45, XO karyotype. The AMEL, DXS6854, DX6803, X22, XHPRT, DXYS218 markers are present with one peak. The SRY marker does not show any signal, while TAF9B marker shows up with 1:0.5 ratio, the GT10_STS47 signal is also missing.

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Fig. 2. Electrophoretogram of a fetus having 47, XXY karyotype. The X chromosome specific markers are present in 1:0.5 ratio related to AMEL gene markers.

chorionic villus sampling [17]. The karyogram shows all structural and numerical chromosomal abnormalities, while the reporting time varies between 2 and 33 weeks. To overcome this long reporting time molecular biological methods were introduced to detect the most common trisomies (FISH and QF-PCR). Both are suitable for the reliable detection of these common trisomies [10,11,18–21]. We started using QF-PCR 16 years ago at Semmelweis University [10,11]. Our in house developed multiplex PCR system is suitable for detection of the most common autosomal trisomies, but not for all sex chromosomal trisomies. It is not applicable for the diagnosis of Turner and Triple X syndromes. It was similar in other laboratories

until special markers were introduced to diagnose these sex chromosome aneuploidies. We received 20.173 amniotic fluid samples in the last 8 years; from these we detected 274 autosomal and 50 sex chromosome trisomies. We checked the reliability of a commercial kit for the detection of X and Y chromosome related trisomies in our clinical sample collection. Based on our results the kit is suitable for the reliable and fast detection of these aneuploidies. The advantage of this kit is that it involves special markers. The number of the sex chromosomal aneuploidies was high in our clinical sample collection. We found 19 samples with 45, XO, 17 samples with 47, XXY, 9 samples with 47, XXX and 5 samples with 47, XYY karyotypes. All observed sex chromosomal

Fig. 3. Electrophoretogram of a fetus with 47, XYY karyotype (double Y syndrome 47, XYY). The AMEL gene marker is present in 1:2 ratio.

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Fig. 4. Electrophoretogram of a sample with 47, XXX karyotype. All X chromosome specific markers are present in 1:1:1, 1:0.5 or 2:1 ratio.

aneuploidies detected with cytogenetic analyses were diagnosed with QF-PCR and DNA fragment analyses. A recent study perform in the Chinese population published their results on the detection of sex chromosome aneuploidies in their population using their own developed kit. They confirmed the usability of their test by involving only 50 healthy controls and 5–5 cases only having sex chromosome aneuploidies [23]. We prefer to use those commercially available kits which have the IVD certificate. According to our study the applied commercial kit is suitable for fast and reliable detection of the sex chromosome related trisomies. These QF-PCR tests detect 99.9% of clinically significant chromosome abnormalities [22]. There could be problems related to maternal cell contamination in bloodstained samples, mosaics and chimeric samples. The detection rate of these is different in each QF-PCR system, but it varies between 5 and 20%. The observed frequencies in our collection in the different sex chromosome abnormalities are in accordance with the earlier published incidence (1/400). Besides the revolution of the non-invasive prenatal detection (NIPD) of the most common trisomies using free DNA from the maternal blood and applying the massive parallel sequencing method, QF-PCR still has the place in prenatal diagnostic methods in the future. Confirming the NIPD results which show the presence of trisomy, we have to prove it by prenatal testing from amniotic fluid cells or chorionic villus samples. Karyotyping has a long turnaround time, and to get fast and reliable results the QF-PCR is the method of choice to confirm the NIPD results. The number of invasive prenatal testing will be decreasing in the future.

5. Conclusions Recently introduced quantitative fluorescent PCR and DNA fragment analysis kit are suitable for the detection of all sex chromosome aneuploidies.

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