QF-PCR Results Research Articles

Utilizing Fetal MRI and Prenatal Medical Exome Sequencing for Prenatal Diagnosis of Kinked Brainstem: A Case Series

Objective: Utilizing fetal MRI and prenatal Medical Exome Sequencing (pMES) for prenatal diagnosis in fetuses exhibiting a kinked or 'z-shaped' brainstem. Methods: A retrospective analysis was conducted on cases of brainstem kinking identified on antenatal imaging between January 2013 and December 2022. Systematic review was performed on clinical data from these cases, encompassing maternal demographics, indications for invasive prenatal diagnosis, ultrasound findings, SNP-array results, Trio-Medical Exome Sequencing (Trio-MES) results, QF-PCR results, pregnancy outcomes and follow-ups. Results: Two cases of fetuses presenting with a "kinked brainstem," along with other CNS abnormalities in fetal MRI were identified using Trio pMES. Prenatal phenotype features observed in our case series include "Z" shaped brainstem, hydrocephalus, hyperechogenic kidneys and generalized skin edema, cobblestone lissencephaly, cerebellar dysplasia, cerebral arachnoid cyst. Trio pMES identified a homozygous variant at c.815_816del (p.Leu272ArgfsTer117) in the FKRP gene for Patient 1. For Patient 2, a de novo heterozygous variant c.848A>G (p.H283R) was detected in the TUBA1A gene. Both variants were predicted to be likely pathogenic or pathogenic. Conclusion: A kinked or "z-shaped" brainstem is a finding that can be more easily delineated on fetal MRI compared to fetal ultrasound, indicating an underlying dystroglycanopathy (Walker-Warburg phenotype) or tubulinopathy. Prenatal exome sequencing can aid in achieving early prenatal genetic diagnosis for cases of "kinked brainstem". Moreover, our study expands its scope by incorporating additional features such as hyperechogenic kidneys and generalized skin edema into the prenatal findings of Walker-Warburg syndrome.

Prenatal phenotype features and genetic etiology of the Williams-Beuren syndrome and literature review.

To share our experience on prenatal diagnosis of Williams-Beuren syndrome(WBS) and to improve the awareness, diagnosis, and intrauterine monitoring of the fetuses of this disease. The study retrospectively evaluated 14 cases of WBS diagnosed prenatally by single nucleotide polymorphism array (SNP-array). Clinical data from these cases were systematically reviewed, including maternal demographics, indications for invasive prenatal diagnosis, ultrasound findings, SNP-array results, trio-medical exome sequencing (Trio-MES) results, QF-PCR results, pregnancy outcomes and follow-ups. A total of 14 fetuses were diagnosed with WBS and their prenatal phenotypes were assessed retrospectively. In our case series, the most common ultrasound features were intrauterine growth retardation (IUGR), congenital cardiovascular defects, abnormal fetal placental doppler indices, thickened nuchal translucency(NT) and polyhydramnios. Other less common ultrasound features include fetal hydrops, hydroderma, bilateral pleural effusion, subependymal cysts, etc. Parental chromosome analysis was performed in seven pairs of parents, and all the deletions on chromosome 7q11.23 were de novo. Prenatal ultrasound features of WBS cases are highly variable, with IUGR, cardiovascular abnormalities and abnormal fetal placental doppler indices, being the most common intrauterine phenotypes. Our case series expand the intrauterine phenotypes of WBS, including cardiovascular abnormalities right aortic arch(RAA) combined with persistent right umbilical vein(PRUV) and elevated the ratio of end-systolic peak flow velocity to end-diastonic peak flow velocity(S/D). In the meantime, with the decrease in the cost of the next-generation sequencing, the method may become widely used in prenatal diagnosis in the near future.

All-in-one whole exome sequencing strategy with simultaneous copy number variant, single nucleotide variant and absence-of-heterozygosity analysis in fetuses with structural ultrasound anomalies: A 1-year experience.

We performed a 1-year evaluation of a novel strategy of simultaneously analyzing single nucleotide variants (SNVs), copy number variants (CNVs) and copy-number-neutral Absence-of-Heterozygosity from Whole Exome Sequencing (WES) data for prenatal diagnosis of fetuses with ultrasound (US) anomalies and a non-causative QF-PCR result. After invasive diagnostics, whole exome parent-offspring trio-sequencing with exome-wide CNV analysis was performed in pregnancies with fetal US anomalies and a non-causative QF-PCR result (WES-CNV). On request, additional SNV-analysis, restricted to (the) requested gene panel(s) only (with the option of whole exome SNV-analysis afterward) was performed simultaneously (WES-CNV/SNV) or as rapid SNV-re-analysis, following a normal CNV analysis. In total, 415 prenatal samples were included. Following a non-causative QF-PCR result, WES-CNV analysis was initially requested for 74.3% of the chorionic villus (CV) samples and 45% of the amniotic fluid (AF) samples. In case WES-CNV analysis did not reveal a causative aberration, SNV-re-analysis was requested in 41.7% of the CV samples and 17.5% of the AF samples. All initial analyses could be finished within 2weeks after sampling. For SNV-re-analysis during pregnancy, turn-around-times (TATs) varied between one and 8days. We show a highly efficient all-in-one WES-based strategy, with short TATs, and the option of rapid SNV-re-analysis after a normal CNV result.

Prenatal diagnosis of Williams-Beuren syndrome by ultrasound and chromosomal microarray analysis

BackgroundThere are a few literature reports of prenatal ultrasound manifestations of Williams-Beuren syndrome. We aimed to explore the prenatal diagnosis of Williams-Beuren syndrome by ultrasound and chromosomal microarray analysis and describe the prenatal ultrasound performance of this syndrome.MethodsIn this retrospective study, we reported eight cases of Williams-Beuren syndrome diagnosed at our prenatal diagnostic center from 2016 to 2021. We systematically reviewed clinical data from these cases, including indications for invasive testing, sonographic findings, QF-PCR results, chromosomal microarray analysis results, and pregnancy outcomes.ResultsIn this study, the common ultrasound features were ventricular septal defect (37.5%), intrauterine growth retardation (25%), and aortic coarctation (25%). Moreover, all patients were found to have a common deletion in the Williams-Beuren syndrome chromosome region at the 7q11.23 locus, which contained the elastin gene. Deletion sizes ranged from 1.42 to 2.07 Mb. Seven parents asked for termination of pregnancy, and one patient was lost to follow-up.ConclusionsThis study is the most extensive prenatal study using chromosomal microarray analysis technology for detailed molecular analysis of Williams-Beuren syndrome cases. We reported three cases combined with first-reported ultrasound manifestations. Case 1 was concomitant with multicystic dysplastic kidney and duodenal atresia combined with case 3. Notably, case 4 was combined with multiple cardiovascular malformations: Tetralogy of Fallot, right aortic arch, and supravalvar aortic stenosis. These manifestations expand the intrauterine ultrasound phenotype of Williams-Beuren syndrome in previous literature reports.

PCR vs karyotype for CVS and amniocentesis—the experience at one tertiary fetal medicine unit

PurposeDespite the rise of non-invasive screening tests for fetal aneuploidy, invasive testing during pregnancy remains the definitive diagnostic tool for fetal genetic anomalies. Results are rapidly available with polymerase chain reaction (PCR) tests, but cases have been reported whereby initial results were not confirmed after pregnancy termination and the fetal karyotype was ultimately normal. We sought to examine the potential discordance between PCR and karyotype for fetal aneuploidy.MethodsThe results from all amniocentesis and CVS tests performed over a 6-year period in a large tertiary level fetal medicine unit were reviewed. The results of PCR and karyotype were recorded and discrepancies examined. Pregnancy outcomes were also recorded.ResultsA total of 1222 invasive tests were performed (716 amniocentesis and 506 CVS). Within the cohort having amniocentesis, 11 had discrepant results (normal QF-PCR result but with a subsequent abnormal karyotype). There was 1 case among this group which QF-PCR should have identified. Within the CVS group, 7 patients had discrepant results. All had a diploid QF-PCR and would not have been identified as abnormal by it.ConclusionPCR can be reliably used to determine aneuploidy of chromosomes 13, 18, and 21. However, in cases of sex chromosome aneuploidy, its performance is less reliable and warrants waiting for a complete karyotype. Given such discordance, we advise waiting for karyotype for all invasive tests performed in the presence of a normal ultrasound before advising a patient of a diploid QF-PCR result or potentially terminating a normal pregnancy.

Diagnostic Outcomes for Genetic Testing of 54 Genes in Pregnancy Loss Using Array CGH Method: A Two-Year Retrospective Study

Objective: The current study aimed to retrospectively evaluate different diag- nostic approaches for the array genetic analysis of the cases from all trimester fetal loss in the medical genetics clinic between 2016 and 2017. The Quantitati- ve Fluorescent Polymerase Chain Reaction(QFPCR) test was performed on 50 samples, and aneuploidy was detected in 11 samples as a result of the test, and the array-CGH was performed when 39 QF-PCR resulted in normal test results. Under this purpose, we aimed to analyze and determine the possible copy num- ber variation(CNV), gene deletions, and/or duplications involved in embryonic cell division, tissue differentiation, intended.Materials and methods: DNA isolation from cases of this retrospective study was completed using the PureLink Genomic DNA isolation kit. DNA samples were then genoty - ped for molecular etiological reasons by oligonücleotid microarray -CGH method (aCGH , 60 K ISCA design , Agilent , Germany ). Hybridized probe correlations of the case and reference DNAs were evaluated with databases (Database of Genomic Variants Analysis ) used in genomic variation analysis in terms of 54 functional genes CNVs associated with intrauterine losses.Results: CNV was detected in 30 (77%) of 39 fetal samples analyzed within the scope of the research. Fifty-five percent of CNVs were found to be duplication (55%) and forty-five percent were deletions (45%). As a result of the evaluation, deletion was detected in 19 (35%) of 54 genes, duplication was detected in 26 (48%), while in 3 (6%) both deletion and duplication were detected. Although CNV detected in autosomal chromosomes (chromosome 1, 2, 3, 4, 5, 7, 8, 10, 12, 13, 14, 15 and 20), CNV was established the most common in X chromosome. In our study, CNVs associated with COX7B, ZIC1, MECP2, FMR1, HOXD13, JAG1, MSX2, NEXN, and SIX3 genes were found to be more frequent in terms of fetal loss etiology.Conclusions: Based on our experience, the array-CGH method can be used to investigate the etiology of the normal results of QF-PCR in cases of fetal loss. The array CGH method will be preferred more and more due to the ease of application and the data obtained. When we look at the literature, it is seen that there is not enough research on array CGH about fetal loss and more studies are needed to increase the experience in this field.

P20.06: The experience with whole‐genome array as a first‐line cytogenetic test in invasive prenatal diagnosis

Comparative Genomic Hybridisation (CGH) array represents a molecular cytogenetic method able to detect submicroscopic copy number variants with a high resolution of hundreds of kb. The aim of this study was to assess the contribution array CGH as a first-line cytogenetic method in routine prenatal diagnosis. This was a prospective study. The workflow for prenatal samples was framed as follows. After the invasive procedure, rapid QF-PCR targeted on Trisomy 13, 18 and 21 was performed. In the case of normal QF-PCR results, we carried on with array CGH. The workflow was introduced in January 2015 for chorionic villi sampling (CVS) and in October 2016 for amniocentesis (AMC). SurePrint G3 Unrestricted Human CGH ISCA v2 8x60K Microarray (Agilent, USA) was used on uncultured CVS/AMC samples. The array resolution was set to ≥ 500 kb in fetuses with normal ultrasound findings and to ≥ 300 kb in fetuses with abnormal ultrasound finding with the exception of smaller deletions/duplication hitting causal genes. In total, 2981 invasive procedures (771 CVS, 2210 AMC) were performed since the beginning of the study. QF-PCR detected Trisomy 13, 18 or 21 in 245 cases (201 (26.1%) CVS, 44 (2.0%) AMC). Therefore, array CGH was conducted in the rest 2703 cases (553 CVS, 2150 AMC). Out of these, 2648 cases had normal results and 55 cases (28 (5.1%) CVS, 27 (1.3%) AMC) showed significant pathological findings. The aberrations were of a size more than 10 Mb in 8 cases, whereas in 47 cases (23 (4.2%) CVS, 24 (1.1%) AMC) the aberration size was below 10Mb. These 47 (1.7%) cases would be thus undetected by conventional karyotyping. Typically, the array results were available up to 7 days after the procedure. Array CGH represents a powerful tool which can be used instead conventional karyotyping in the prenatal diagnosis. The advantage is that it detects 1.7% submicroscopic pathologic deletions/duplications which would be undetected using conventional karyotyping and the results are available typically up to 7 days. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Rapid Prenatal Diagnosis of Chromosomal Aneuploidies Using Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR)

Introduction: Prenatal diagnosis plays an important role in determining the health of fetus, which is why rapid diagnostic tests are so important. One of these quick diagnostic tests is quantitative fluorescent-polymerase chain (QF- PCR) technique. This technique employed to detect chromosomal aneuploidies (13, 18, 21, X and Y). In this method, there is no need for cell culturing. QF- PCR is rapid, suitable for automation, low cost and faster turnaround time. Material and Methods: In this study, 260 patients were studied, and analyzed by 26 short tandem repeat (STR) markers for detection of chromosomal abnormalities. The QF-PCR assay was performed by Devyser kit (Sweden). First DNA was extracted, and then PCR was performed, PCR products were run on capillary electrophoresis system, and finally analyzed by the Genemarker Software. Results: The results of QF-PCR were as follows: trisomy 21 (6 cases), 18 (3 cases), triploidy (1 case), klinefelter syndrome (1 case), without suspicion of any mosaicism. Conclusion: Prenatal diagnostic outcomes give early results, thus reducing parental anxiety and increasing clinical management of a high risk pregnancy.

MG-104 Fetal jugular lymph sacs – what is the significance?: Abstract MG-104 Table 1

Fetal jugular lymph sacs are accumulations of lymphatic fluid in the anterolateral region of the fetal neck. They may be isolated or occur in association with other structural anomalies and in some cases are presenting with an increased nuchal translucency or cystic hygroma. Their significance and the guidelines for the prenatal investigation when identified in the course of routine ultrasonography in pregnancy are still controversial. Our protocol for the investigation of these cases includes microarray analysis and DNA analysis for the Noonan syndrome panel. We report our experience with ten cases of prenatally diagnosed isolated fetal jugular neck cysts identified in the last 5 years (Table 1). In 10 cases identified by us in the last 5 years, 50% had a pathogenic mutation in a Noonan syndrome Panel, one had trisomy 21 and none had abnormality on microarray analysis (in the case with T21 we stopped the analysis following the QF-PCR results). However, all cases with abnormal results had NT > 3.5 mm or cystic hygroma. This study suggests that the identification of fetal jugular lymph sacs with an increased NT/cystic hygroma is associated with an increased incidence of Noonan syndrome and its related disorders and should instigate mutation analysis of the Noonan syndrome panel.

The microdeletion/microduplication profiles in spontaneously aborted fetal materials: Double blind results of QF-PCR and MLPA techniques

The microdeletion/microduplication profiles in spontaneously aborted fetal materials: Double blind results of QF-PCR and MLPA techniques

Rapid diagnosis of the most common fetal aneuploidies with the QF-PCR method--a study of 100 cases

The aim of the study was to assess whether commercial kit QF-PCR can be used as the only method for rapic prenatal dia gnosis of chromosomes 13, 18, 21, X and Y aneuploidies, omitting cell culture and complete cyt6genetik analysis of fetal chromosomes. DNA from amniocytes (94 cases) and trophoblast cells (6 cases) was analyzed witt QF-PCR according to the manufacturer's protocol. The obtained products were separated using ABI 310 Genetic Analyzer and the resulting data were analyzed using GeneMarker software. The results of QF-PCR were obtained in 95 out of 100 cases (95%). Abnormalities were found in 28 casea (29.5%). All these results were confirmed in subsequent cytogenetic analysis. Normal results were obtained in 62 patients (70.5%). However in that group, we found three chromosomal aberrations other than those analyzed b3 QF-PCR. Additionally two abnormal and three normal karyotypes were found in patients with inconclusive QF-POF results. QF-PCR is a fast and reliable tool for chromosomal aneuploidy analysis and can be used as the only method without a full analysis of the karyotype, but only in cases of suspected fetal 13, 18, 21 trisomy or numerica aberrations of X chromosome. In other cases, fetal karyotype analysis from cells obtained after cell culture should be offered to the patient.

PS-317 Qf-pcr As A Stand-alone Test For Diagnosis Of Aneuploides In Pre And Postnatal Samples: An Indian Report

Down syndrome is the most common aneuploides seen in live born babies with the prevalence of 1 in 1000 followed by other trisomies. The burden of aneuploides can be reduced with new molecular cytogenetic technology which helps in early intervention and genetic counselling. As our centre is being a referral centre for all genetic disorders, we receive a high risk couples related to chromosomal abnormalities as having one affected child. Since we are providing QF-PCR as a stand-alone test for postnatal diagnosis, the same methodology was extrapolated for prenatal diagnosis. Initially, 500 postnatal samples (Blood in EDTA vial) and 240 amniotic fluid samples were received for analysis of chromosomal aneuploidies from various part of the country. The DNA was extracted with QIAamp DNA mini kit by Qiagen. QF-PCR was carried out with the following markers D21S1411, D21S11, D21S1435, D21S1412, AMEL, SRY, D18S535, D18S391, D13S258, D13S634 and XHPRT, X22 whose heterozygosity have been studied. We observed 100% concordance with the clinical diagnosis as well as cytogenetic analysis of postnatal samples. With these results we went for prenatal services were chromosomal studies are very common for suspected Down syndrome pregnancies. Out of 240 pregnancies studied, 2% (5) was of Trisomy 21 and a single case of Trisomy 18 was identified, these results were also reconfirmed with karyotyping results. Results of present investigation reassure QF-PCR as stand-alone test for high risk pregnancies. This is the first study from India that tested the in-house developed multiplex QF-PCR for post and prenatal diagnosis.

Chromosome Aberrations Identified by Cytogenetic Analysis of the First Two Clones of Cultured Amniotic Fluid Cells Compared with QF-PCR Results

We report on our experience of studying amniotic fluid cells by cytogenetic analysis (CA) of the first 2 clones. We investigated the incidence and types of chromosome aberrations detected by CA of 196 amniocenteses performed on pregnant women at high risk. Of these cases, 178 were analysed by QF-PCR (risk group A). The results were compared with the data obtained by CA of 1,263 amniocenteses carried out in patients with other indications. QF-PCR was used to investigate 1,030 of these cases (risk group B). The combined average turnaround time for a CA result of the first 2 clones in both risk groups was within 9 ± 2 days. The final CA results (≥6 clones) were obtained within 12 ± 4 days. In risk group A, CA was not possible in 2 cases due to cell culture failure. The foetal karyotype was abnormal in 13.8% of the cases by CA of ≥6 clones and in 13.5% of the cases by QF-PCR. Together, CA of ≥6 clones and QF-PCR detected chromosome aberrations in 14.8% of the cases. With the exception of 2 cases of in vitro culture failure and 1 case with low gonosomal mosaicism, CA of the first 2 clones detected all cases with chromosome aberrations. Five cases with clinically significant chromosome aberrations were not detected by QF-PCR. In risk group B, the foetal karyotype was found to be abnormal in 2.2% of the cases by CA of ≥6 clones and in 1.0% of the cases by QF-PCR. Together, CA of ≥6 clones and QF-PCR revealed chromosome aberrations in 2.2% of the cases. With the exception of 1 case with low gonosomal mosaicism, CA of the first 2 clones detected all other cases with chromosome aberrations. The majority of these cases were inherited chromosome aberrations. Eighteen cases with chromosome aberrations were not detected by QF-PCR. Based on our results, CA of ≥6 clones, together with QF-PCR as a first test, should be performed in all prenatal cases with abnormal ultrasound findings. In pregnancies with other indications, CA of the first 2 clones alone is sufficient to identify all clinically significant (and inherited) chromosome aberrations.

“If it helps …” the use of microarray technology in prenatal testing: Patient and partners reflections

The objective was to gain insight into the experiences of women and their partners diagnosed with a fetal abnormality on prenatal ultrasound examination and receiving genetic testing including microarray. Twenty-five semi-structured interviews were performed with women +/- their partners after receiving the results of prenatal genetic testing. Framework analysis was performed to elicit themes and subthemes. Five main themes were recognized; diagnosis, genetic testing, family and support, reflections of the treatment received and emotions. Our results showed that women recall being told about QFPCR for trisomy 13, 18, and 21 but often no further testing. Women expected the conventional karyotype and microarray result would be normal following a normal QFPCR result. There were frequent misconceptions by couples regarding aspects of counseling/testing. Communication of variants of unknown (clinical) significance (VOUS) presents a particularly difficult challenge. Good clear communication by health care professionals is paramount. When counseling women and their partners for fetal chromosomal testing it should be reinforced that although the most common, trisomy 13, 18, and 21 only account for some of the chromosomal changes resulting in abnormal scan findings. Couples should have literature to take home summarizing scan anomalies and reinforcing information about genetic testing.

PF.30 The Use of Microarray Technology in Prenatal Testing, Patient and Partners Reflections

In the prenatal setting, women are offered fetal karyotyping for reasons including an abnormality on ultrasound scan. Full, conventional karyotyping has been available since the 1960s but is being superseded...

Prenatal diagnosis – a comparison of rapid testing by QF-PCR versus full karyotyping in the Scottish population

Current health policy encourages the use of rapid trisomy testing rather than full karyotype analysis. We compared the results of QF-PCR with full karyotyping via a retrospective audit. Between 01/11/09...

Non‐targeted whole genome 250K SNP array analysis as replacement for karyotyping in fetuses with structural ultrasound anomalies: evaluation of a one‐year experience

We evaluated both clinical and laboratory aspects of our new strategy offering quantitative fluorescence (QF)-PCR followed by non-targeted whole genome 250K single-nucleotide polymorphism array analysis instead of routine karyotyping for prenatal diagnosis of fetuses with structural anomalies. Upon the detection of structural fetal anomalies, parents were offered a choice between QF-PCR and 250K single-nucleotide polymorphism array analysis (QF/array) or QF-PCR and routine karyotyping (QF/karyo). Two hundred twenty fetal samples were included. In 153/220 cases (70%), QF/array analysis was requested. In 35/153 (23%), an abnormal QF-PCR result was found. The remaining samples were analyzed by array, which revealed clinically relevant aberrations, including two known microdeletions, in 5/118 cases. Inherited copy number variants were detected in 11/118 fetuses, copy number variants with uncertain clinical relevance in 3/118 and homozygous stretches in 2/118. In 67/220 (30%) fetuses, QF/karyo was requested: 23/67 (34%) were abnormal with QF-PCR, and in 3/67, an abnormal karyotype was found. Even though QF/array does not reveal a high percentage of submicroscopic aberrations in fetuses with unselected structural anomalies, it is preferred over QF/karyo, as it provides a whole genome scan at high resolution, without additional tests needed and with a low chance on findings not related to the ultrasound anomalies.

QF-PCR as a Rapid Technique for Routine Prenatal Diagnosis of Fetal Aneuploidies

Background: The most common chromosomal abnormalities are aneuploidies of chromosome 21, 18, 13, X and Y. Prenatal diagnosis of fetal aneuploidies is routinely done by traditional cytogenetic culture, a major drawback of this technique is the long period required to reach a diagnosis. In this study we evaluated the QF-PCR as a rapid technique for prenatal diagnosis of common aneuploidies Method: This work was carried out on Sixty amniotic fluid samples taken from patients with one or more of the following indications: Advanced maternal age (3 case), abnormal biochemical markers (6 cases), abnormal ultrasound (12 cases) or previous history of abnormal child (39 cases). Each sample was tested by QF-PCR and traditional cytogenetic. Aneuploidy screenings were performed amplifying four STRs on chromosomes 21, 18, 13, two pseudoautosomal,one X linked, as well as the AMXY and SRY; markers were distributed in two multiplex QFPCR assays (S1 and S2) in order to reduce the risk of sample mishandling. Results: All the QF-PCR results were successful, while there was two culture failures, only one of them was repeated. No discrepancy was seen between the results of both techniques. Fifty six samples showed normal patterns, three sample showed trisomy 21, successfully detected by both techniques and one sample showed normal pattern by QF-PCR but could not be compared to the cytogenetics due to culture failure, the pregnancy outcome of this case was a normal baby. Conclusion: Our study concluded that QF-PCR is a reliable technique for prenatal diagnosis of the common chromosomal aneuploidies.

Incidence of placental mosaicism leading to discrepant results between QF‐PCR and karyotyping in 22,825 chorionic villus samples

To review the frequency and analyse the origin of completely discrepant results observed between QF-PCR and karyotyping in chorionic villus samples (CVS) as a result of placental mosaicism. Also, to assess QF-PCR results for biallelic or triallelic patterns and determine their significance. Between May 2002 and December 2009, 22 825 CVS were received at TDL Genetics for processing by QF-PCR and karyotype. The QF-PCR and karyotype data were compared to determine the incidence of discrepant results. Of the 22 825 samples received, 22 779 (99.8%) gave concordant results between the PCR and karyotype, and 46 samples (0.2%) gave discordant results. Of these discrepant cases, 5 displayed triallelic peaks and 41 displayed biallelic peaks. All discordant results are due to the presence of placental mosaicism, a known limitation of using this sample type for prenatal diagnosis. This retrospective study of placental mosaicism in CVS is the largest single centre study to date and provides a figure for the occurrence of completely discrepant results between QF-PCR and karyotype due to placental mosaicism. This study also demonstrates that the presence of triallelic peaks at QF-PCR is not sufficient to exclude the presence of placental mosaicism.

Detection of trisomy 21 by quantitative fluorescent PCR in clinical samples undergoing prenatal diagnosis for hereditary hearing loss

To establish the genetic test technique of trisomy 21 concurrently conducts with prenatal diagnosis for hereditary hearing loss. Fifty-four pregnant women who underwent prenatal diagnosis for hearing loss of their fetuses in Chinese People's Liberation Army General Hospital from March 2009 to May 2010 were enrolled in this study. All probands from the deaf families have confirmed the causative mutation for hearing loss in Genetic Testing Center in Chinese People's Liberation Army General Hospital. The mean age of 54 pregnant women is 31 years at pregnancy of 18 - 26 weeks, 5 cases > pregnancy of 23 weeks, 9 cases ≥ 35 years. All subjects did not conduct the serologic tests for trisomy 21 before. Fifteen to twenty ml amniotic fluid was drawn from 49 cases at pregnancy of 18 - 23 weeks and 5 cases > pregnancy of 23 weeks. One to two ml umbilical blood was drawn from 5 cases > pregnancy of 23 weeks. For 9 cases ≥ 35 years, amniotic fluid cell culture and karyotyping analysis were conducted concurrently. A multiple quantitative fluorescent (QF) PCR and six microsatellite markers were applied to diagnosis trisomy 21. The samples with peaks of 1:1:1 or 2:1 at two microsatellite markers can be diagnosed as trisomy 21. (1) Fifty-four fetuses were successfully conducted prenatal genetic diagnosis for hearing loss (included GJB2 and SLC26A4). Ten fetuses copied the exactly same genotypes as the probands. The other 44 cases fetuses did not copy the same genotypes as the probands and won't develop hearing loss. The hearing test showed normal hearing for the neonates. (2) All the 54 fetuses were excluded of trisomy 21 by QF-PCR and were verified after birth. Five fetuses with advanced maternal age were performed karyotyping analysis and showed normal. The diagnostic results of QF-PCR can be obtained in 1-3 days without misdiagnosed. QF-PCR is an efficient, rapid and accurate technique for detection of trisomy 21 without increasing sample amount. It can be used for fetuses who were undertaken hearing loss gene test or other prenatal gene test.