Galjaard, R-J.H.

Application of SNP array for rapid prenatal diagnosis: Implementation, genetic counselling and diagnostic flow (Article)

2011-12-01T00:00:00Z

We report on the validation and implementation of the HumanCytoSNP-12 array (Illumina) (HCS) in prenatal diagnosis. In total, 64 samples were used to validate the Illumina platform (20 with a known (sub) microscopic chromosome abnormality, 5 with known maternal cell contamination (MCC) and 39 normal control samples). There were no false-positive or false-negative results. In addition to the diagnostic possibilities of arrayCGH, the HCS allows detection of regions of homozygosity (ROH), triploidy and helps recognising MCC. Moreover, in two cases of MCC, a deletion was correctly detected. Furthermore we found out that only about 50 ng of DNA is required, which allows a reporting time of only 3 days. We also present a prospective pilot study of 61 fetuses with ultrasound abnormalities and a normal karyotype tested with HCS. In 4 out of 61 (6.5%) fetuses, a clinically relevant abnormality was detected. We designed and present pre-test genetic counselling information on categories of possible test outcomes. On the basis of this information, about 90% of the parents chose to be informed about adverse health outcomes of their future child at infancy and childhood, and 55% also about outcomes at an adult stage. The latter issue regarding the right of the future child itself to decide whether or not to know this information needs to be addressed.

XCI in preimplantation mouse and human embryos: first there is remodelling... (Article)

2011-06-07T00:00:00Z

Female eutherians silence one of their X chromosomes to accomplish an equal dose of X-linked gene expression compared with males. The mouse is the most widely used animal model in XCI research and has proven to be of great significance for understanding the complex mechanism of X-linked dosage compensation. Although the basic principles of XCI are similar in mouse and humans, differences exist in the timing of XCI initiation, the genetic elements involved in XCI regulation and the form of XCI in specific tissues. Therefore, the mouse has its limitations as a model to understand early human XCI and analysis of human tissues is required. In this review, we describe these differences with respect to initiation of XCI in human and mouse preimplantation embryos, the extra-embryonic tissues and the in vitro model of the epiblast: the embryonic stem cells.

Economic evaluation of multiplex ligation-dependent probe amplification and karyotyping in prenatal diagnosis: a cost-minimization analysis (Article)

2011-05-19T00:00:00Z

Purpose: To assess the cost-effectiveness of Multiplex Ligation-dependent Probe Amplification (MLPA, P095 kit) compared to karyotyping. Methods: A cost-minimization analysis alongside a nationwide prospective clinical study of 4,585 women undergoing amniocentesis on behalf of their age (≥36 years), an increased risk following first trimester prenatal screening or parental anxiety. Results: Diagnostic accuracy of MLPA (P095 kit) was comparable to karyotyping (1.0 95% CI 0.999-1.0). Health-related quality of life did not differ between the strategies (summary physical health: mean difference 0.31, p = 0.82; summary mental health: mean difference 1.91, p = 0.22). Short-term costs were lower for MLPA: mean difference €315.68 (bootstrap 95% CI €315.63-315.74; -44.4%). The long-term costs were slightly higher for MLPA: mean difference €76.42 (bootstrap 95% CI €71.32-81.52; +8.6%). Total costs were on average €240.13 (bootstrap 95% CI €235.02-245.23; -14.9%) lower in favor of MLPA. Cost differences were sensitive to proportion of terminated pregnancies, sample throughput, individual choice and performance of tests in one laboratory, but not to failure rate or the exclusion of polluted samples. Conclusion: From an economic perspective, MLPA is the preferred prenatal diagnostic strategy in women who undergo amniocentesis on behalf of their age, following prenatal screening or parental anxiety.

Defective deacetylation of histone 4 K12 in human oocytes is associated with advanced maternal age and chromosome misalignment (Article)

2011-05-01T00:00:00Z

Background: Chromosome segregation errors during human oocyte meiosis are associated with low fertility in humans and the incidence of these errors increases with advancing maternal age. Studies of mitosis and meiosis suggest that defective remodeling of chromatin plays a causative role in aneuploidy. We analyzed the histone deacetylation pattern during the final stages of human oocyte maturation to investigate whether defective epigenetic regulation of chromatin remodeling in human oocytes is related to maternal age and leads to segregation errors.MethodsHuman surplus oocytes of different meiotic maturation stages [germinal vesicle (GV), metaphase (M)I and MII] were collected from standard IVF/ICSI treatments. Oocytes were analyzed for acetylation of different lysines of histone 4 (H4K5, H4K8, H4K12 and H4K16) and for α-tubulin. ResultsHuman GV oocytes had an intense staining of the chromatin for all four histone 4 lysine acetylations. MI and MII stage oocytes showed either normal deacetylation or various amounts of defective histone deacetylation. Residual H4K12 acetylation was more frequently found in oocytes obtained from older women, with a significant correlation between defective deacetylation and maternal age (r 0.185, P 0.007). Eighty-eight percent of the oocytes with residual acetylation had misaligned chromosomes, whereas only 33 of the oocytes that showed correct deacetylated chromatin had misaligned chromosomes (P < 0.001). Conclusions We conclude that defective deacetylation during human female meiosis increases with maternal age and is correlated with misaligned chromosomes. As chromosome misalignment predisposes to segregation errors, our data imply that defective regulation of histone deacetylation could be an important factor in age-related aneuploidy.

Multiplex ligation dependent probe amplification (MLPA) for rapid distinction between unique sequence positive and negative marker chromosomes in prenatal diagnosis (Article)

2011-01-18T00:00:00Z

Background: Small supernumerary marker chromosomes (sSMC) are extra structurally abnormal chromosomes that cannot be unambiguously identified with conventional chromosome banding techniques. These marker chromosomes may cause an abnormal phenotype or be harmless depending on different factors such as genetic content, chromosomal origin and level of mosaicism. When a sSMC is found during prenatal diagnosis, the main question is whether the sSMC contains euchromatin since in most cases this will lead to phenotypic abnormalities. We present the use of Multiplex Ligation Dependent probe Amplification (MLPA) for rapid distinction between non-euchromatic and euchromatic sSMC. Results: 29 well-defined sSMC found during prenatal diagnosis were retrospectively investigated with MLPA with the SALSA MLPA centromere kits P181 and P182 as well as with the SALSA MLPA telomere kits P036B and P070 (MRC Holland BV, Amsterdam, The Netherlands). All unique-sequence positive sSMC were correctly identified with MLPA, whereas the unique-sequence negative sSMC had normal MLPA results. Conclusions: Although different techniques exist for identification of sSMC, we show that MLPA is a valuable adjunctive tool for rapidly distinguishing between unique-sequence positive and negative sSMC. In case of positive MLPA results, genetic microarray analysis or, if not available, targeted FISH can be applied for further identification and determination of the exact breakpoints, which is important for prediction of the fetal phenotype. In case of a negative MLPA result, which means that the sSMC most probably does not contain genes, the parents can already be reassured and parental karyotyping can be initiated to assess the heritability. In the mean time, FISH techniques are needed for determination of the chromosomal origin.

Omphalocele: Comparison of outcome following prenatal or postnatal diagnosis (Article)

2010-12-01T00:00:00Z

Objectives To assess the impact of prenatal compared with postnatal diagnosis on outcome for liveborn infants with an isolated or with a non-isolated omphalocele. Methods This was a retrospective analysis of 101 prenatally and 45 postnatally diagnosed cases of omphalocele. Cases were collected from the ultrasound database of the Division of Obstetrics and Prenatal Medicine and the patient database of the Department of Pediatric Surgery. Results Following confirmation at delivery or autopsy, prenatally diagnosed omphaloceles included 21 isolated cases, 44 non-isolated cases with a normal karyotype and 36 non-isolated cases with an abnormal karyotype. Of the prenatally diagnosed apparently isolated cases (n = 31), 12 (39%; 95% CI, 22-58%) revealed associated anomalies after delivery. Liveborn infants with an isolated omphalocele had significantly worse short-term morbidity following prenatal diagnosis (n = 14) compared with diagnosis at birth (n = 29), having a lower gestational age at delivery, lower Apgar scores, longer duration of ventilation and parenteral nutrition, more readmissions and a longer hospital stay. The prenatally diagnosed subset contained more infants with a giant omphalocele (9/14 vs. 3/29, P = 0.001) and liver herniation (8/14 vs. 6/29, P = 0.02). The outcome of liveborn infants with a non-isolated omphalocele diagnosed prenatally (n = 17) was not different from that of those diagnosed at birth (n = 16), except for a greater need for ventilation and parenteral nutrition in the prenatal subset. Conclusion When counseling patients with a prenatal diagnosis of isolated omphalocele, it is important to remember that over one third could turn out to have associated anomalies. Liveborn infants with an isolated omphalocele detected prenatally have worse short-term morbidity than do cases detected at birth. Those with non-isolated omphaloceles detected prenatally have an increased need for ventilation and parenteral nutrition compared with those detected at birth. Copyright

Ethnic differences in participation in prenatal screening for Down syndrome: A register-based study (Article)

2010-10-01T00:00:00Z

Objective: To assess ethnic differences in participation in prenatal screening for Down syndrome in the Netherlands. Methods: Participation in prenatal screening was assessed for the period 1 January 2009 to 1 July 2009 in a defined postal code area in the southwest of the Netherlands. Data on ethnic origin, socio-economic background and age of participants in prenatal screening were obtained from the Medical Diagnostic Centre and the Department of Clinical Genetics. Population data were obtained from Statistics Netherlands. Logistic regression models were used to assess ethnic differences in participation, adjusted for socio-economic and age differences. Results: The overall participation in prenatal screening was 3865 out of 15 093 (26%). Participation was 28% among Dutch women, 15% among those from Turkish ethnic origin, 8% among those from North-African origin, 15% among those from Aruban/Antillean origin and 26% among women from Surinamese origin. Conclusions: Compared to Dutch women, those from Turkish, North-African, Aruban/Antillean and other non-Western ethnic origin were less likely to participate in screening. It was unexpected that women from Surinamese origin equally participated. It should be further investigated to what extent participation and non-participation in these various ethnic groups was based on informed decision-making.

Ethnic and socio-economic differences in uptake of prenatal diagnostic tests for Down's syndrome (Article)

2010-08-01T00:00:00Z

Objective: The objective of this study was to assess ethnic and socio-economic differences in the uptake of maternal age-based prenatal diagnostic testing for Down's syndrome by amniocentesis or chorionic villus sampling. Study design: The study population consisted of 12,340 women aged 36 years or over, who lived in a geographically defined region in the Southwest of The Netherlands and who gave birth to a live born infant in the period 2000-2004. Data were obtained from the Department of Clinical Genetics Erasmus MC and Statistics Netherlands. Logistic regression analyses were done to assess ethnic and socio-economic differences in uptake. Results: The overall uptake of prenatal diagnostic tests was 28.5%. Women of Turkish and Caribbean origin participated in prenatal diagnostic tests equally or more often than Dutch women. Women of North-African origin and women from low socio-economic background had a lower uptake than others. Ethnic differences in uptake could not be attributed to differences in socio-economic background. Conclusions: Uptake of prenatal diagnostic tests for Down's syndrome in The Netherlands was low and varied among ethnic and socio-economic groups of advanced maternal age. The finding that women of Turkish and Caribbean origin participated in prenatal diagnostic tests equally or more often than Dutch women was unexpected. The low uptake among Dutch women may be related to the Dutch pregnancy culture. The finding that women of North-African origin and women from low socio-economic background had a lower uptake may be related to barriers in access to prenatal diagnostic tests.

Comparison of multiplex ligation-dependent probe amplification and karyotyping in prenatal diagnosis (Article)

2010-02-01T00:00:00Z

Abstract. OBJECTIVE: To estimate whether multiplex ligation-dependent probe amplification (MLPA), a molecular technique used for detecting the most common chromosomal aneuploidies, is comparable with karyotyping for the detection of aneuploidies of chromosomes X, Y, 13, 18, and 21 in routine clinical practice and to estimate the costs differences of both techniques. METHODS: In this prospective, nationwide cohort study, we consecutively included 4,585 women who had an amniocentesis because of their age (36 years or older), increased risk after prenatal screening, or maternal anxiety. Amniotic fluid samples were tested independently with both MLPA and karyotyping. The primary outcome was diagnostic accuracy of MLPA to detect aneuploidies of chromosomes X, Y, 13, 18, and 21. Secondary outcome measures were turnaround time for test results and costs. A sample size was calculated using a critical noninferiority margin of 0.002; therefore, at least 4,497 paired test results were needed (one-sided alpha 0.05, power 0.90). RESULTS: Diagnostic accuracy of MLPA was 1.0 (95% confidence interval [CI] 0.99-1.0), sensitivity was 100% (95% CI 0.96-1.0) and specificity was 100% (95% CI 0.999-1.0). Diagnostic accuracy of MLPA was statistically similar (noninferior) to that of karyotyping (P<.001). In 75 cases, MLPA failed (1.6%); karyotyping failed once (0.02%). Compared with karyotyping, MLPA shortened the waiting time by 14.5 days (P<.001, 95% CI 14.3-14.6) and cost less (-47, P<.001). CONCLUSION: In routine clinical practice, diagnostic accuracy of MLPA for detection of trisomies X, Y, 13, 18, and 21 is comparable with that of karyotyping, and it reduces waiting time at lower costs.

High-resolution identification of balanced and complex chromosomal rearrangements by 4C technology (Article)

2009-10-15T00:00:00Z

Balanced chromosomal rearrangements can cause disease, but techniques for their rapid and accurate identification are missing. Here we demonstrate that chromatin conformation capture on chip (4C) technology can be used to screen large genomic regions for balanced and complex inversions and translocations at high resolution. The 4C technique can be used to detect breakpoints also in repetitive DNA sequences as it uniquely relies on capturing genomic fragments across the breakpoint. Using 4C, we uncovered LMO3 as a potentially leukemogenic translocation partner of TRB@. We developed multiplex 4C to simultaneously screen for translocation partners of multiple selected loci. We identified unsuspected translocations and complex rearrangements. Furthermore, using 4C we detected translocations even in small subpopulations of cells. This strategy opens avenues for the rapid fine-mapping of cytogenetically identified translocations and inversions, and the efficient screening for balanced rearrangements near candidate loci, even when rearrangements exist only in subpopulations of cells.

X chromosome inactivation is initiated in human preimplantation embryos (Article)

2009-06-01T00:00:00Z

X chromosome inactivation (XCI) is the mammalian mechanism that compensates for the difference in gene dosage between XX females and XY males. Genetic and epigenetic regulatory mechanisms induce transcriptional silencing of one X chromosome in female cells. In mouse embryos, XCI is initiated at the preimplantation stage following early whole-genome activation. It is widely thought that human embryos do not employ XCI prior to implantation. Here, we show that female preimplantation embryos have a progressive accumulation of XIST RNA on one of the two X chromosomes, starting around the 8-cell stage. XIST RNA accumulates at the morula and blastocyst stages and is associated with transcriptional silencing of the XIST-coated chromosomal region. These findings indicate that XCI is initiated in female human preimplantation-stage embryos and suggest that preimplantation dosage compensation is evolutionarily conserved in placental mammals.

Rapid aneuploidy detection with multiplex ligation-dependent probe amplification: A prospective study of 4000 amniotic fluid samples (Article)

2009-01-01T00:00:00Z

The introduction of prenatal screening requires rapid high-throughput diagnosis of common aneuploidies. Multiplex ligation-dependent probe amplification (MLPA) allows for quick, easily automated multiplex testing of these aneuploidies in one polymerase chain reaction. We performed a large prospective study using MLPA on 4000 amniotic fluid (AF) samples including all indications and compared its value to karyotyping and fluorescence in situ hybridization (FISH). MLPA can reliably determine common aneuploidies with 100% sensitivity and 100% specificity. Moreover, some mosaic cases and structural chromosome aberrations were detected as well. In cases of a male fetus, triploidies can be detected by an aberrant pattern of probe signals, which mimics maternal cell contamination (MCC). Macroscopic blood contamination was encountered in 3.2% of the AF samples. In 20% of these samples, an MLPA pattern was found consistent with MCC, although there were no false negatives of the most common aneuploidies. As the vast majority of inconclusive results (1.7%) is due to potential MCC, we designed a protocol in which we determine whether MLPA can be performed on blood-contaminated AF samples by testing if blood is of fetal origin. Then, the number of inconclusive results could be theoretically reduced to 0.05%. We propose an alternative interpretation of relative probe signals for rapid aneuploidy diagnosis (RAD). We discuss the value of MLPA for the detection of (submicroscopic) structural chromosome anomalies. MLPA is a reliable method that can replace FISH and could be used as a stand-alone test for RAD instead of karyotyping.

Embryonic delay in growth and development related to confined placental trisomy 16 mosaicism, diagnosed by I-Space Virtual Reality (Article)

2008-11-01T00:00:00Z

Objective: To demonstrate the use of a novel three-dimensional (3D) virtual reality (VR) system in the visualization of first trimester growth and development in a case of confined placental trisomy 16 mosaicism (CPM+16). Design: Case report. Setting: Prospective study on first trimester growth using a 3D VR system. Patient(s): A 34-year-old gravida 1, para 0 was seen weekly in the first trimester for 3D ultrasound examinations. Intervention(s): Chorionic villus sampling was performed because of an enlarged nuchal translucency (NT) measurement and low pregnancy-associated plasma protein-A levels, followed by amniocentesis. Result(s): Amniocentesis revealed a CPM+16. On two-dimensional (2D) and 3D ultrasound no structural anomalies were found with normal fetal Dopplers. Growth remained below the 2.3 percentile. At 37 weeks, a female child of 2010 g (<2.5 percentile) was born. After birth, growth climbed to the 50th percentile in the first 2 months. Conclusion(s): The I-Space VR system provided information about phenotypes not obtainable by standard 2D ultrasound. In this case, the delay in growth and development could be observed very early in pregnancy. Since first trimester screening programs are still improving and becoming even more important, systems such as the I-Space open a new era for in vivo studies on the physiologic and pathologic processes involved in embryogenesis.

An incomplete trisomy 3 rescue resulting in a marker chromosome and UPD(3) - Difficulties in interpretation (Article)

2008-10-01T00:00:00Z

False positive FISH diagnosis of monosomy X in uncultured amniotic fluid cells due to a chromosome Y deletion (Article)

2008-09-01T00:00:00Z

Genome-wide oligonucleotide-based array comparative genome hybridization analysis of non-isolated congenital diaphragmatic hernia (Article)

2007-02-15T00:00:00Z

Non-isolated congenital diaphragmatic hernia (CDH+) is a severe birth defect that is often caused by de novo chromosomal anomalies. In this report, we use genome-wide oligonucleotide-based array comparative genome hybridization (aCGH) followed by rapid real-time quantitative PCR analysis to identify, confirm and map chromosomal anomalies in a cohort of 26 CDH+ patients. One hundred and five putative copy number changes were identified by aCGH in our cohort of CDH+ patients. Sixty-one of these changes (58%) had been previously described in normal controls. Twenty of the remaining 44 changes (45%) were confirmed by quantitative real-time PCR or standard cytogenetic techniques. These changes included de novo chromosomal abnormalities in five of the 26 patients (19%), two of whom had previously normal G-banded chromosome analyses. Data from these patients provide evidence for the existence of CDH-related genes on chromosomes 2q37, 6p22-25 and 14q, and refine the CDH minimal deleted region on 15q26 to an interval that contains COUP - TFII and only eight other known genes. Although COUP - TFII is likely to play a role in the development of CDH in patients with 15q26 deletions, we did not find COUP - TFII mutations in 73 CDH samples. We conclude that the combination of oligonucleotide-based aCGH and quantitative real-time PCR is an effective method of identifying, confirming and mapping clinically relevant copy number changes in patients with CDH+. This method is more sensitive than G-banded chromosome analysis and may find wide application in screening patients with congenital anomalies.

Congenital diaphragmatic hernia associated with duplication of 11q23-qter. (Article)

2006-07-15T00:00:00Z

Congenital diaphragmatic hernia (CDH) is a relatively common birth defect with a high mortality. Although little is known about its etiology, there is increasing evidence for a strong genetic contribution. Both numerical and structural chromosomal abnormalities have been described in patients with CDH. Partial trisomy 11q and partial trisomy 22 associated with the common t(11;22) has been reported in several cases of CDH. It has been assumed that the diaphragmatic defect seen in these individuals was primarily due to duplication of material from chromosome 22q11. However, in this report we describe a family with a t(11;12) in which one of two brothers with partial trisomy 11q has a left sided posterolateral CDH. This is the second case of CDH in partial trisomy 11q due to an unbalanced translocation other than t(11;22). Using array-based comparative genomic hybridization and fluorescent in situ hybridization, we mapped the breakpoints in both brothers and their mother who is a balanced translocation carrier. Our results suggest that duplication of one or more genes on a approximately 19 Mb region of 11q23.3-qter predisposes to the development of CDH. These effects may be the primary cause of CDH in individuals t(11;22) or may be additive to effects from the duplication of chromosome 22 material. We also conclude that the partial trisomy 11q syndrome has a variable phenotype and that CDH should be added to the spectrum of anomalies that can be present in this syndrome.

Congenital diaphragmatic hernia and chromosome 15q26: determination of a candidate region by use of fluorescent in situ hybridization and array-based comparative genomic hybridization (Article)

2005-01-01T00:00:00Z

Congenital diaphragmatic hernia (CDH) has an incidence of 1 in 3,000 births and a high mortality rate (33%-58%). Multifactorial inheritance, teratogenic agents, and genetic abnormalities have all been suggested as possible etiologic factors. To define candidate regions for CDH, we analyzed cytogenetic data collected on 200 CDH cases, of which 7% and 5% showed numerical and structural abnormalities, respectively. This study focused on the most frequent structural anomaly found: a deletion on chromosome 15q. We analyzed material from three of our patients and from four previously published patients with CDH and a 15q deletion. By using array-based comparative genomic hybridization and fluorescent in situ hybridization to determine the boundaries of the deletions and by including data from two individuals with terminal 15q deletions but without CDH, we were able to exclude a substantial portion of the telomeric region from the genetic etiology of this disorder. Moreover, one patient with CDH harbored a small interstitial deletion. Together, these findings allowed us to define a minimal deletion region of approximately 5 Mb at chromosome 15q26.1-26.2. The region contains four known genes, of which two--NR2F2 and CHD2--are particularly intriguing gene candidates for CDH.

Mapping Studies of Congenital Limb Anomalies (Doctoral Thesis)

2003-09-24T00:00:00Z

Clinical features and neuroimaging of PARK7-linked Parkinsonism (Article)

2003-07-01T00:00:00Z

We recently reported linkage to chromosome 1p36 (the PARK7-locus) in a family with early-onset parkinsonism. Linkage to this locus has since been confirmed in an independent data set. We describe clinical and neuroimaging features of the 4 patients in the original PARK7-linked kindred. Age at onset of parkinsonism varied from 27 to 40 years. Clinical progression was slow, and response to dopaminergic therapy good. The clinical spectrum ranged from mild hypokinesia and rigidity, to severe parkinsonism with levodopa-induced dyskinesias and motor fluctuation. Three of four patients with PARK7-linked parkinsonism exhibited psychiatric disturbances. Structural neuroimaging was unremarkable, but functional imaging of the brain, carried out in 3 patients, showed significant evidence for a presynaptic dopamine deficit, and assessment of cerebral glucose metabolism, as carried out in 1 patient, showed possible cerebellar involvement.

Park7, a novel locus for autosomal recessive early-onset parkinsonism, on chromosome 1p36 (Article)

2001-01-01T00:00:00Z

Although the role of genetic factors in the origin of Parkinson disease has long been disputed, several genes involved in autosomal dominant and recessive forms of the disease have been localized. Mutations associated with early-onset autosomal recessive parkinsonism have been identified in the Parkin gene, and recently a second gene, PARK6, involved in early-onset recessive parkinsonism was localized on chromosome 1p35-36. We identified a family segregating early-onset parkinsonism with multiple consanguinity loops in a genetically isolated population. Homozygosity mapping resulted in significant evidence for linkage on chromosome 1p36. Multipoint linkage analysis using MAPMAKER-HOMOZ generated a maximum LOD-score of 4.3, with nine markers spanning a disease haplotype of 16 cM. On the basis of several recombination events, the region defining the disease haplotype can be clearly separated, by > or =25 cM, from the more centromeric PARK6 locus on chromosome 1p35-36. Therefore, we conclude that we have identified on chromosome 1 a second locus, PARK7, involved in autosomal recessive, early-onset parkinsonism.