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Background: Noninvasive prenatal detection of fetal subchromosomal copy number aberrations (CNAs) can be achieved through massively parallel sequencing of maternal plasma DNA. However, when a mother herself is a carrier of a CNA, one cannot discern if her fetus has inherited the CNA. In addition, false-positive results would become more prevalent when more subchromosomal regions are analyzed., Methods: We used a strategy that combined count- and size-based analyses of maternal plasma DNA for the detection of fetal subchromosomal CNAs in 7 target regions for 10 test cases., Results: For the 5 cases in which CNAs were present only in the fetus, the size-based approach confirmed the aberrations detected by the count-based approach. For the 5 cases in which the mother herself carried an aberration, we successfully deduced that 3 of the fetuses had inherited the aberrations and that the other 2 fetuses had not inherited the aberrations. No false positives were observed in this cohort., Conclusions: Combined count- and size-based analysis of maternal plasma DNA permits the noninvasive elucidation of whether a fetus has inherited a CNA from its mother who herself is a carrier of the CNA. This strategy has the potential to improve the diagnostic specificity of noninvasive prenatal testing., (© 2016 American Association for Clinical Chemistry.)

Objective: To evaluate the clinical impact of nationwide implementation of genome-wide non-invasive prenatal testing (NIPT) in pregnancies at increased risk for fetal trisomies 21, 18 and 13 (TRIDENT study)., Method: Women with elevated risk based on first trimester combined testing (FCT ≥ 1:200) or medical history, not advanced maternal age alone, were offered NIPT as contingent screening test, performed by Dutch University Medical laboratories. We analyzed uptake, test performance, redraw/failure rate, turn-around time and pregnancy outcome., Results: Between 1 April and 1 September 2014, 1413/23 232 (6%) women received a high-risk FCT result. Of these, 1211 (85.7%) chose NIPT. One hundred seventy-nine women had NIPT based on medical history. In total, 1386/1390 (99.7%) women received a result, 6 (0.4%) after redraw. Mean turn-around time was 14 days. Follow-up was available in 1376 (99.0%) pregnancies. NIPT correctly predicted 37/38 (97.4%) trisomies 21, 18 or 13 (29/30, 4/4 and 4/4 respectively); 5/1376 (0.4%) cases proved to be false positives: trisomies 21 (n = 2), 18 (n = 1) and 13 (n = 2). Estimated reduction in invasive testing was 62%., Conclusion: Introduction of NIPT in the Dutch National healthcare-funded Prenatal Screening Program resulted in high uptake and a vast reduction of invasive testing. Our study supports offering NIPT to pregnant women at increased risk for fetal trisomy. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd., (© 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.)

Objective: To evaluate preferences and decision-making among high-risk pregnant women offered a choice between Non-Invasive Prenatal Testing (NIPT), invasive testing or no further testing., Methods: Nationwide implementation study (TRIDENT) offering NIPT as contingent screening test for women at increased risk for fetal aneuploidy based on first-trimester combined testing (>1:200) or medical history. A questionnaire was completed after counseling assessing knowledge, attitudes and participation following the Multidimensional Measure of Informed Choice., Results: A total of 1091/1253 (87%) women completed the questionnaire. Of these, 1053 (96.5%) underwent NIPT, 37 (3.4%) invasive testing and 1 (0.1%) declined testing. 91.7% preferred NIPT because of test safety. Overall, 77.9% made an informed choice, 89.8% had sufficient knowledge and 90.5% had positive attitudes towards NIPT. Women with intermediate (odds ratio (OR) = 3.51[1.70-7.22], p < 0.001) or high educational level (OR = 4.36[2.22-8.54], p < 0.001) and women with adequate health literacy (OR = 2.60[1.36-4.95], p = 0.004) were more likely to make an informed choice. Informed choice was associated with less decisional conflict and less anxiety (p < 0.001). Intention to terminate the pregnancy for Down syndrome was higher among women undergoing invasive testing (86.5%) compared to those undergoing NIPT (58.4%) (p < 0.001)., Conclusions: The majority of women had sufficient knowledge and made an informed choice. Continuous attention for counseling is required, especially for low-educated and less health-literate women. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd., (© 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.)

Early in pregnancy women and their partners face the complex decision on whether or not to participate in prenatal testing for fetal chromosomal abnormalities. Several studies show that the majority of pregnant women currently do not make informed decisions regarding prenatal testing. As the range of prenatal tests is expanding due to the development of new techniques such as non-invasive prenatal testing (NIPT), autonomous reproductive decision-making is increasingly challenging. In this study, a randomised controlled trial was conducted to evaluate the effect of a web-based multimedia decision aid on decision-making regarding prenatal testing. The decision aid provided both written and audiovisual information on prenatal tests currently available, that is, prenatal screening by first-trimester combined testing, NIPT and invasive diagnostic testing through chorionic villus sampling or amniocentesis. Furthermore, it contained values clarification exercises encouraging pregnant women to reflect on the potential harms and benefits of having prenatal tests performed. The use of the decision aid improved informed decision-making regarding prenatal testing. Of pregnant women allocated to the intervention group (n=130) 82.3% made an informed choice compared with 66.4% of women in the control group (n=131), P=0.004. As the vast majority of pregnant women made decisions consistent with their attitudes towards having prenatal testing performed, this improvement in informed decision-making could be attributed mainly to an increase in decision-relevant knowledge. This study shows that the implementation of a web-based multimedia decision aid directly facilitates the ultimate goal of prenatal testing for fetal chromosomal abnormalities, which is enabling informed autonomous reproductive choice.

Objective: To validate Illumina's two-channel NextSeq 500 sequencing system for noninvasive prenatal testing (NIPT) of fetal whole chromosome and partial aberrations., Methods: A total of 162 plasma samples, previously sequenced for NIPT on a SOLiD 5500xl platform, were sequenced on the NextSeq 500 using 75-bp single-end sequencing, followed by analysis using the WISECONDOR algorithm., Results: For whole chromosome aneuploidy detection, all samples were classified correctly (in total 3× T13, 3× T18, 8× T21 and 145× euploid). Three partial aberrations (36-Mb terminal loss of 5p, 14-Mb gain on 18p and 33-Mb terminal loss of 13q) were also correctly identified. Fetal fractions in 34 male samples sequenced on both the SOLiD 5500xl and NextSeq 500 platform showed no significant difference. To test robustness, two sample sets, containing both euploid and aneuploid samples, were sequenced on different NextSeq 500 machines, revealing identical results. With unchanged laboratory flow, the NIPT turnaround time could be reduced from 15-16 calendar days to 7-8 calendar days, after switching from the SOLiD 5500xl to the NextSeq 500 platform., Conclusions: The NextSeq 500 platform can be used for NIPT to detect both whole and partial chromosome aberrations. It has fast turnaround times and is suitable for mid-sized laboratories., (© 2016 John Wiley & Sons, Ltd.)

Background: Noninvasive genetic tests that use cell-free fetal DNA (cffDNA) are used increasingly in prenatal care. A low amount of cffDNA can have detrimental effects on the reliability of these tests. A marker to confirm the presence of fetal nucleic acids is therefore required that is universally applicable and easy to incorporate., Methods: We developed a novel multiplex, single-tube, noninvasive fetal sex determination assay by combining amplification of AMELY cffDNA with one-step reverse transcription (RT)-PCR of trophoblast-derived cell-free RNA (cfRNA), which functions as a sex-independent fetoplacental marker. We tested plasma samples from 75 pregnant women in duplicate in a blinded fashion. The fetus was considered to be male in the case of a positive result for AMELY and cfRNA amplification in both RT-PCRs. The fetus was considered to be female in the case of negative AMELY and positive cfRNA result in both RT-PCRs. In other cases, the test was repeated. We compared the results with invasive prenatal testing and pregnancy outcomes., Results: The AMELY cffDNA amplification and cfRNA result was unambiguous and identical in duplicate in 71 of 75 plasma samples (95%). Four samples (5%) required an extra replicate because of an absent fetoplacental marker. Thereafter, fetal sex was correctly determined in all 75 plasma samples., Conclusions: Amplification of trophoblast-derived cfRNA is a reliable marker for the confirmation of the presence of fetoplacentally derived nucleic acids in noninvasive fetal sex determination., (© 2015 American Association for Clinical Chemistry.)

Objective: This study evaluates pregnant women's and healthcare professionals' preferences regarding specific prenatal screening and diagnostic test characteristics., Method: A discrete choice experiment was developed to assess preferences for prenatal tests that differed in seven attributes: minimal gestational age, time to test results, level of information, detection rate, false positive rate, miscarriage risk and costs., Results: The questionnaire was completed by 596 (70.2%) pregnant women and 297 (51.7%) healthcare professionals, of whom 507 (85.1%) and 283 (95.3%), respectively, were included in further analyses as their choice behavior indicated prenatal testing was an option to them. Comparison of results showed differences in relative importance attached to attributes, further reflected by differences in willingness to trade between attributes. Pregnant women are willing to accept a less accurate test to obtain more information on fetal chromosomal status or to exclude the risk of procedure-related miscarriage. Healthcare professionals consider level of information and miscarriage risk to be most important as well but put more emphasis on timing and accuracy., Conclusion: Pregnant women and healthcare professionals differ significantly in their preferences regarding prenatal test characteristics. Healthcare professionals should take these differences into consideration when counseling pregnant women on prenatal testing., (© 2015 John Wiley & Sons, Ltd.)

Noninvasive prenatal testing (NIPT) for fetal aneuploidies using cell-free fetal DNA in maternal plasma has revolutionized the field of prenatal care and methods using massively parallel sequencing are now being implemented almost worldwide. Substantial progress has been made from initially testing for (an)euploidies of chromosomes 13, 18 and 21, to testing for sex chromosome (an)euploidies, additional autosomal aneuploidies as well as partial deletions and duplications genome-wide. Although NIPT is associated with significantly reduced risks for the fetus in comparison to existing invasive prenatal diagnostic methods, it presents several implementation challenges. Here, we review key issues potentially influencing NIPT and illustrate them using both data from literature and in-house data.

In its successful annual cycle of controversies and debates, the International Society of Prenatal Diagnosis and Therapy once again addressed non-invasive prenatal testing (NIPT) by following up on the 2013 controversy, 'Should non-invasive DNA testing be the standard screening test for Down syndrome in all pregnant women'? with the proposition, 'NIPT for chromosomel abnormalities should be offered to women with low a priori risk'., (© 2014 John Wiley & Sons, Ltd.)

Objective: Non-invasive prenatal testing (NIPT) using cell-free fetal DNA in maternal plasma has been developed for the detection of fetal aneuploidy. Clinical trials have shown high sensitivity and specificity for trisomy 21 (T21) in both high-risk and average-risk populations. Although its great potential for prenatal medicine is evident, more information regarding the consequences of implementing NIPT in a national programme for prenatal screening is required., Study Design: A decision-analytic model was developed to compare costs and outcomes of current clinical practice in The Netherlands using conventional screening only, with two alternatives: implementing NIPT as an optional secondary screening test for those pregnancies complicated by a high risk for T21, and implementing NIPT as primary screening test, replacing conventional screening. Probability estimates were derived from a systematic review of international literature. Costs were determined from a health-care perspective. Data were analysed to obtain outcomes, total costs, relative costs and incremental cost-effectiveness ratios (ICERs) for the different strategies. Sensitivity analysis was used to assess the impact of assumptions on model results., Results: Implementing NIPT as an optional secondary, or as primary screening test will increase T21 detection rate by 36% (from 46.8% to 63.5%) and 54% (from 46.8% to 72.0%), simultaneously decreasing the average risk of procedure-related miscarriage by 44% (from 0.0168% to 0.0094% per pregnant woman) and 62% (from 0.0168% to 0.0064% per pregnant woman), respectively. None of the strategies clearly dominated: current clinical practice is the least costly, whereas implementing NIPT will cause total costs of the programme to increase by 21% (from €257.09 to €311.74 per pregnant woman), leading to an ICER of k€94 per detected case of T21, when utilised as an optional secondary screening test and by 157% (from €257.09 to €660.94 per pregnant woman), leading to an ICER of k€460 per detected case of T21, when utilised as primary screening test. However, implementing NIPT as triage test did result in the lowest expected relative costs per case of T21 diagnosed (k€141)., Conclusion: NIPT should be implemented in national health care as an optional secondary screening test for those pregnancies complicated by a high risk for T21., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: The aim of this study was to evaluate the best diagnostic approach for the genetic analysis of samples from first, second and third trimester intrauterine fetal deaths (IUFDs). We examined a total of 417 IUFD samples from fetuses with and without congenital anomalies. On 414 samples, karyotyping (N = 46) and/or rapid aneuploidy testing by QF-PCR (N = 371) was performed). One hundred sixty eight samples with a normal test result were subsequently tested by genome wide Single Nucleotide Polymorphism (SNP) array analysis. Three samples were only analyzed by array., Results: In 50 (12.0%) samples an aneuploidy was detected by QF-PCR and/or karyotyping, representing 47.1% of first, 13.2% of second and 3.4% of third trimester pregnancies. Karyotyping and QF-PCR failed in 4 (8.7%) and 7 (1.9%) samples, respectively, concerning mostly contaminated amniotic fluid samples from third trimester pregnancies.Clinically relevant aberrations were identified in 4.2% (all fetuses with malformations) of the 168 samples tested by SNP array. Inherited copy number variants (CNVs) were detected in 5.4% and 8.9% showed CNVs of unknown clinical relevance as parental inheritance could not be studied yet. In a sample from a fetus suspect for Meckel-Grüber syndrome, the genotype information from the SNP array revealed various stretches of homozygosity, including one stretch encompassing the CEP290 gene. Subsequent CEP290 mutation analysis revealed a homozygous, pathogenic mutation in this gene., Conclusions: Based on our experience we recommend QF-PCR as the first-line test in IUFD samples of first and second trimester pregnancies to exclude aneuploidy before performing array analysis. The chance to detect aneuploidy in third trimester pregnancies is relatively low and therefore array analysis can be performed as a first-tier test. A tissue sample, instead of amniotic fluid, is preferred because of a higher success rate in testing.We emphasize the need for analysis of parental samples whenever a rare, unique CNV is detected to allow for better interpretation of such findings and to improve future pregnancy management. Furthermore, we illustrate the strength of SNP arrays for genotype analysis, even though we realize it is crucial to have detailed phenotypic information to make optimal use of the genotype data in finding candidate recessive genes that may be related to the fetal phenotype.

Objectives: Circulating cell-free fetal DNA (ccffDNA) in maternal plasma is an attractive source for noninvasive prenatal testing (NIPT). The amount of total cell-free DNA significantly increases 24h after venipuncture, leading to a relative decrease of the ccffDNA fraction in the blood sample. In this study, we evaluated the downstream effects of extended processing times on the reliability of aneuploidy detection by massively parallel sequencing (MPS)., Design and Methods: Whole blood from pregnant women carrying normal and trisomy 21 (T21) fetuses was collected in regular EDTA anti-coagulated tubes and processed within 6h, 24 and 48h after venipuncture. Samples of all three different time points were further analyzed by MPS using Z-score calculation and the percentage of ccffDNA based on X-chromosome reads., Results: Both T21 samples were correctly identified as such at all time-points. However, after 48h, a higher deviation in Z-scores was noticed. Even though the percentage of ccffDNA in a plasma sample has been shown previously to significantly decrease 24h after venipuncture, the percentages based on MPS results did not show a significant decrease after 6, 24 or 48h., Conclusions: The quality and quantity of ccffDNA extracted from plasma samples processed up to 24h after venipuncture are sufficiently high for reliable downstream NIPT analysis by MPS. Furthermore, we show that it is important to determine the percentage of ccffDNA in the fraction of the sample that is actually used for NIPT, as downstream procedures might influence the fetal or maternal fraction., (© 2013.)

In recent studies on prenatal testing for Noonan syndrome (NS) in fetuses with an increased nuchal translucency (NT) and a normal karyotype, mutations have been reported in 9-16% of cases. In this study, DNA of 75 fetuses with a normal karyotype and abnormal ultrasound findings was tested in a diagnostic setting for mutations in (a subset of) the four most commonly mutated NS genes. A de novo mutation in either PTPN11, KRAS or RAF1 was detected in 13 fetuses (17.3%). Ultrasound findings were increased NT, distended jugular lymphatic sacs (JLS), hydrothorax, renal anomalies, polyhydramnios, cystic hygroma, cardiac anomalies, hydrops fetalis and ascites. A second group, consisting of anonymized DNA of 60 other fetuses with sonographic abnormalities, was tested for mutations in 10 NS genes. In this group, five possible pathogenic mutations have been identified (in PTPN11 (n=2), RAF1, BRAF and MAP2K1 (each n=1)). We recommend prenatal testing of PTPN11, KRAS and RAF1 in pregnancies with an increased NT and at least one of the following additional features: polyhydramnios, hydrops fetalis, renal anomalies, distended JLS, hydrothorax, cardiac anomalies, cystic hygroma and ascites. If possible, mutation analysis of BRAF and MAP2K1 should be considered.

The goal to noninvasively detect fetal aneuploidies using circulating cell-free fetal DNA in the maternal plasma seems to be achieved by the use of massively parallel sequencing (MPS). To date, different MPS approaches exist, all aiming to deliver reliable results in a cost effective manner. The most widely used approach is the whole genome MPS method, in which sequencing is performed on maternal plasma to determine the presence of a fetal trisomy. To reduce costs targeted approaches, only analyzing loci from the chromosome(s) of interest has been developed. This review summarizes the different MPS approaches, their benefits and limitations and discusses the implications for future noninvasive prenatal testing., (© 2013 John Wiley & Sons, Ltd.)

Blood plasma of pregnant women contains circulating cell-free fetal DNA (ccffDNA), originating from the placenta. The use of this DNA for non-invasive detection of fetal aneuploidies using massively parallel sequencing (MPS)-by-synthesis has been proven previously. Sequence performance may, however, depend on the MPS platform and therefore we have explored the possibility for multiplex MPS-by-ligation, using the Applied Biosystems SOLiD(™) 4 system. DNA isolated from plasma samples from 52 pregnant women, carrying normal or aneuploid fetuses, was sequenced in multiplex runs of 4, 8 or 16 samples simultaneously. The sequence reads were mapped to the human reference genome and quantified according to their genomic location. In case of a fetal aneuploidy, the number of reads of the aberrant chromosome is expected to be higher or lower than in normal reference samples. To statistically determine this, Z-scores per chromosome were calculated as described previously, with thresholds for aneuploidies set at > +3.0 and < -3.0 for chromosomal over- or underrepresentation, respectively. All samples from fetal aneuploidies yielded Z-scores outside the thresholds for the aberrant chromosomes, with no false negative or positive results. Full-blown fetal aneuploidies can thus be reliably detected in maternal plasma using a multiplex MPS-by-ligation approach. Furthermore, the results obtained with a sample from a pregnancy with 45,X in the cytotrophoblastic cell layer and 46,XX in the mesenchymal core cells show that ccffDNA originates from the cytotrophoblastic cell layer. Discrepancies between the genetic constitution of this cell layer and the fetus itself are well known, and therefore, care should be taken when translating results to the fetus itself.

Objective: 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., Methods: 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)., Results: 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., Conclusion: 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., (© 2012 John Wiley & Sons, Ltd.)

As a routine, karyotyping of invasive prenatal samples is performed as an adjunct to referrals for DNA mutation detection and metabolic testing. We performed a retrospective study on 500 samples to assess the diagnostic value of this procedure. These samples included 454 (90.8%) chorionic villus (CV) and 46 (9.2%) amniocenteses specimens. For CV samples karyotyping was based on analyses of both short-term culture (STC) and long-term culture (LTC) cells. Overall, 19 (3.8%) abnormal karyotypes were denoted: four with a common aneuploidy (trisomy 21, 18 and 13), two with a sex chromosomal aneuploidy (Klinefelter syndrome), one with a sex chromosome mosaicism and twelve with various autosome mosaicisms. In four cases a second invasive test was performed because of an abnormal finding in the STC. Taken together, we conclude that STC and LTC karyotyping has resulted in a diagnostic yield of 19 (3.8%) abnormal cases, including 12 cases (2.4%) with an uncertain significance. From a diagnostic point of view, it is desirable to limit uncertain test results as secondary test findings. Therefore, we recommend a more targeted assay, such as e.g. QF-PCR, as a replacement of the STC and to provide parents the autonomy to choose between karyotyping and QF-PCR.

Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.

Improvements in non-invasive screening methods for trisomy 21 (Down syndrome) and other aneuploidies during the first and second trimester of pregnancy have radically changed the indications for prenatal diagnosis over the last decade. Consequently, there was a need for rapid tests for the detection of common chromosome aneuploidies resulting in the development of molecular methods for the rapid, targeted detection of (an)euploidies of the chromosomes 13, 18, 21 and the sex chromosomes. The analysis of large series of prenatal samples has shown that such tests can detect the great majority of chromosome abnormalities in prenatal diagnosis. This resulted in lively discussions on whether conventional karyotyping should remain the standard method for the majority of prenatal cases or can be replaced by rapid tests only. This review gives an overview of different aspects of the three most common tests for rapid, targeted prenatal detection of (an)euploidies, i.e. interphase fluorescence in-situ hybridisation (iFISH), quantitative fluorescent polymerase chain reaction (QF-PCR) and multiplex ligation-dependent probe amplification (MLPA)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Array-based comparative genomic hybridization analysis of genomic DNA was first applied in postnatal diagnosis for patients with intellectual disability (ID) and/or congenital anomalies (CA). Genome-wide single-nucleotide polymorphism (SNP) array analysis was subsequently implemented as the first line diagnostic test for ID/CA patients in our laboratory in 2009, because its diagnostic yield is significantly higher than that of routine cytogenetic analysis. In addition to the detection of copy number variations, the genotype information obtained with SNP array analysis enables the detection of stretches of homozygosity and thereby the possible identification of recessive disease genes, mosaic aneuploidy, or uniparental disomy. Patient-parent (trio) information analysis is used to screen for the presence of any form of uniparental disomy in the patient and can determine the parental origin of a de novo copy number variation. Moreover, the outcome of a genotype analysis is used as a final quality control by ruling out potential sample mismatches due to non-paternity or sample mix-up. SNP array analysis is now also used in our laboratory for patients with disorders for which locus heterogeneity is known (homozygosity pre-screening), in prenatal diagnosis in case of structural ultrasound anomalies, and for patients with leukemia. In this report, we summarize our array findings and experiences in the various diagnostic applications and demonstrate the power of a SNP-based array platform for molecular karyotyping, because it not only significantly improves the diagnostic yield in both constitutional and cancer genome diagnostics, but it also enhances the quality of the diagnostic laboratory workflow., (Copyright © 2011 S. Karger AG, Basel.)

Background: The implementation of microarray analysis in prenatal diagnostics is a topic of discussion, as rare copy number variants with unknown/uncertain clinical consequences are likely to be found. The application of targeted microarrays limits such findings, but the potential disadvantage is that relevant, so far unknown, aberrations might be overlooked. Therefore, we explore the possibilities for the prenatal application of the genome-wide 250k single nucleotide polymorphism array platform., Methods: Affymetrix 250k NspI single nucleotide polymorphism array analysis (Affymetrix, Inc., Santa Clara, California, USA) was performed on DNA from 38 prenatally karyotyped fetuses with ultrasound anomalies. Analyses were performed after termination of pregnancy, intrauterine fetal death or birth on DNA isolated from fetal or neonatal material., Results: Aberrations were detected in 17 of 38 fetuses, 6 of whom with a previously identified chromosomal abnormality and 11 with previously normal or balanced karyotypes. Of the latter, the detected aberration occurred de novo and was considered of clinical relevance in five cases (16%), inherited from a healthy parent in four cases (12%), and de novo yet with unclear clinical relevance in two cases (6%). The clinically relevant abnormalities either were novel copy number variants (n=3) or concerned a uniparental disomy (n=2)., Conclusion: In at least 16% of fetuses with ultrasound anomalies and a normal or balanced karyotype, causal (submicroscopic) aberrations were detected, illustrating the importance of the (careful) implementation of microarray analysis in prenatal diagnosis. The fact that the identified, clinically relevant, aberrations would have gone undetected with most targeted approaches underscores the added value of a genome-wide approach.

Objective: This study aimed to determine the diagnostic application of multiplex ligation-dependent probe amplification (MLPA) as a stand-alone test for targeted detection of common chromosomal aneuploidies (i.e. 13, 18, 21, X and Y) in amniotic fluid cells in routine prenatal clinical practice., Methods: In this evaluation study, the MLPA test using kit P095 was performed on 1000 consecutive amniotic fluid samples and the results obtained were compared with traditional karyotyping (TK), the gold standard., Results: The absolute specificity and sensitivity of the MLPA test were 100%. The test yielded a rapid reporting time: 94% within three working days and 5% within seven working days. The test failure rate was 0.8%. The percentage of abnormalities undetectable using this specific test was 2.4%: abnormal foetal ultrasound (N=9), increased risk first trimester screening (N=2), advanced maternal age (N=3) or other reason for referral (N=10). These abnormalities can be categorised in clinically significant (N=8), clinically uncertain (N=4) and clinically nonsignificant (N=12)., Conclusions: MLPA P095 is suitable as a stand-alone test for the rapid and efficient detection of the most common chromosomal aneuploidies in routine prenatal clinical practice. A flow chart for integrating the MLPA test into the cytogenetic laboratory workflow is presented.

It is known from postnatal diagnosis that imbalances of the subtelomeric regions contribute significantly to idiopathic mental retardation. Consequently, subtelomere screening has been incorporated into the recommendations for the evaluation of individuals with unexplained mental retardation and a normal karyotype. Previous studies suggested that for fetuses with ultrasound abnormalities and a normal karyotype, additional screening for submicroscopic imbalances can be relevant for diagnosis and prognosis. In the present paper, we report the detection of such (subtelomeric) imbalances in three fetuses. Prenatally, the three fetuses presented with ultrasound abnormalities highly suspected of a chromosomal aberration. In two of the fetuses, routine karyotyping showed no aberrations but with MLPA or FISH a small subtelomeric imbalance, that could explain the anomalies, was detected. In the third fetus, a chromosomal abnormality was detected with routine cytogenetic analysis (del(X)(p22.1)), but this abnormality could not explain the ultrasound observations and only with subtelomere screening by MLPA a causative chromosomal aberration was detected. As the three fetuses were already prenatally suspected of a chromosomal aberration, this underlines the potential relevance of subtelomere screening in such fetuses, leading to better clinical diagnosis, prognosis and care. Furthermore, when using MLPA, the analysis can be extended to other regions of known clinical importance.

The deletion 9p syndrome is caused by a constitutional monosomy of part of the short arm of chromosome 9. It is clinically characterized by dysmorphic facial features (trigonocephaly, midface hypoplasia, and long philtrum), hypotonia and mental retardation. Deletion 9p is known to be heterogeneous and exhibits variable deletion sizes. The critical region for a consensus phenotype has been reported to be located within a approximately 4-6 Mb interval on 9p22. In the present study, deletion breakpoints were determined in 13 Dutch patients by applying fluorescence in situ hybridization (FISH) and in some specific cases by array-based comparative genomic hybridization (array CGH). No clear genotype-phenotype correlation could be established for various developmental features. However, we were able to narrow down the critical region for deletion 9p syndrome to approximately 300 kb. A functional candidate gene for trigonocephaly, the CER1 gene, appeared to be located just outside this region. Sequence analysis of this gene in nine additional patients with isolated trigonocephaly did not reveal any pathogenic mutations.

A homozygous mutation of the CNTNAP2 gene has been associated with a syndrome of focal epilepsy, mental retardation, language regression and other neuropsychiatric problems in children of the Old Order Amish community. Here we report genomic rearrangements resulting in haploinsufficiency of the CNTNAP2 gene in association with epilepsy and schizophrenia. Genomic deletions of varying sizes affecting the CNTNAP2 gene were identified in three non-related Caucasian patients. In contrast, we did not observe any dosage variation for this gene in 512 healthy controls. Moreover, this genomic region has not been identified as showing large-scale copy number variation. Our data thus confirm an association of CNTNAP2 to epilepsy outside the Old Order Amish population and suggest that dosage alteration of this gene may lead to a complex phenotype of schizophrenia, epilepsy and cognitive impairment.

Objectives: Introduction of the second-trimester fetal anomaly scan and the decision to offer this scan to every woman in the 18th-22nd week of pregnancy necessitates a re-evaluation of the diagnostic value of the measurement of alpha-fetoprotein (AFP) concentrations in the amniotic fluid (AF) for the detection of neural tube defects (NTDs)., Methods: In this study of 6501 women who underwent amniocentesis, amniotic fluid AFP (AFAFP) concentrations were measured. The women were divided into three categories: group I, without any increased risk of fetal NTD (N = 6188); group II, with an increased risk of fetal NTD (N = 258); and group III, with a clinically diagnosed fetal NTD with known AFAFP concentrations (N = 55)., Results: In 27 women of group I (0.4%), the MoM (multiple of the median) level was > 2.5 times the median AFP concentration for the corresponding gestational age, and in two fetuses this was related to NTD. In two pregnancies of group II (0.8%), an increased AFAFP was related to NTD. In group III, 44 of the 55 (80%) samples had an increased AFAFP., Conclusion: In the near future, it is likely that imaging will replace AFAFP assays for the detection of fetal NTDs because high quality ultrasound imaging will detect NTDs accurately., (Copyright 2007 John Wiley & Sons, Ltd.)

Pure partial duplication or triplication of the proximal part of chromosome 14 has been reported in only 4 patients. Other individuals with a duplication or triplication of this region have additional chromosome imbalances. We present a new case with a supernumerary marker chromosome in all blood cells and in 35% of the cells an additional smaller marker chromosome. Both markers appeared to be derived from chromosome 14 (del(14)(q21.2) in all cells and del(14)(q11.2) in 35% of the cells). This results in a partial duplication of the proximal region of chromosome 14, combined with a mosaic partial triplication of a smaller segment of the same region. In this paper, we compare the clinical features of this case to those of cases from the literature. Although most of the patients from literature were unbalanced translocation carriers, their clinical features were comparable, except from renal abnormalities.

Proximal duplications of the long arm of chromosome 1 are rare and the few patients that have been described in literature have multiple congenital abnormalities and/or mental retardation. The present paper describes the clinical and cytogenetic findings of an adult patient with only mild mental retardation and some minor malformations. The patient carries an inverted duplication of 1q12q21.2.