Opstal, D. van

Another rare prenatal case of post-zygotic mosaic trisomy 17 (Article)

2013-03-22T00:00:00Z

Phenotypic variability of atypical 22q11.2 deletions not including TBX1 (Article)

2012-10-01T00:00:00Z

Interstitial deletions of the chromosome 22q11.2 region are the most common microdeletions in humans. The TBX1 gene is considered to be the major candidate gene for the main features in 22q11.2 deletion syndrome, including congenital heart malformations, (para)thyroid hypoplasia, and craniofacial abnormalities. We report on eight patients with atypical deletions of chromosome 22q11.2. These deletions comprise the distal part of the common 22q11.2 deleted region but do not encompass the TBX1 gene. Ten similar patients with overlapping distal 22q11.2 deletions have been reported previously. The clinical features of these patients are described and compared to those found in the classic 22q11.2 deletion syndrome. We discuss the possible roles of a position effect or haploinsufficiency of distally located genes (e.g., CRKL) in the molecular pathogenesis of the 22q11.2 deletion syndrome.

Prenatally diagnosed submicroscopic familial aberrations at 18p11.32 without phenotypic effect (Article)

2011-12-05T00:00:00Z

Background: Recent development of MLPA (Multiplex-Ligation-dependent Probe Amplification, MRC-Holland) and microarray technology allows detection of a wide range of new submicroscopic abnormalities. Publishing new cases and case reviews associated with both clinical abnormalities and a normal phenotype is of great value. Findings/results. We report on two phenotypically normal foetuses carrying a maternally-inherited interstitial submicroscopic abnormality of chromosome 18p11.32. Both abnormalities were found with the aneuploidy MLPA kit P095 during rapid aneuploidy detection, which was offered along with conventional karyotyping. Foetus 1 and its mother have a 1,7 Mb deletion and foetus 2 and its mother have a 1,9 Mb duplication. In both cases normal babies were born. We used the HumanCytoSNP-12 array of Illumina to visualize the CNVs and map the breakpoints. Conclusions: We suggest that a CNV at 18p11.32 (528,050-2,337,486) may represent a new benign euchromatic variant.

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.

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.

The fate of the mosaic embryo: Chromosomal constitution and development of Day 4, 5 and 8 human embryos (Article)

2010-08-01T00:00:00Z

Background: Post-zygotic chromosome segregation errors are very common in human embryos after in vitro fertilization, resulting in mosaic embryos. However, the significance of mosaicism for the developmental potential of early embryos is unknown. We assessed chromosomal constitution and development of embryos from compaction to the peri-implantation stage. Methods: From 112 cryopreserved Day 4 human embryos donated for research, 21 were immediately fixed and all cells were analysed by fluorescent in situ hybridization (FISH) for chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. The remaining 91 embryos were thawed, with 54 embryos undergoing biopsy of one or two cells which were fixed and analysed by FISH. Biopsied embryos were kept in standard culture conditions for 24 h. Embryos arrested before cavitation (n = 24) were fixed whereas developing Day 5 blastocysts (n = 24) were co-cultured for a further 72 h on an endometrial monolayer followed by fixation. Cell numbers were counted and all nuclei were analysed by FISH. Data from a previous FISH analysis on cryopreserved good-quality Day 5 blastocysts (n = 36) were also included in the present study. Results: FISH analysis was successful for 18 Day 4 fixed embryos and, according to our definition, 83 were mosaic and 11 showed a chaotic chromosomal constitution. FISH analysis of two blastomeres from Day 4 developing embryos showed that 54 were mosaic, 40 were normal and 6 were abnormal. Analysis of Day 4, 5 and 8 whole embryos showed a decrease in incidence of mosaicism over time, from 83 on Day 4 to 42 on Day 8. A significant positive correlation was observed between the total cell number and the percentage of normal cells in developing Day 5 and Day 8 embryos but not in developing Day 4 or embryos arrested before cavitation. Conclusions: These data suggest that both the developmental arrest of a significant proportion of mosaic embryos on Day 4, and the cell death or reduced proliferation of aneuploid cells within an embryo may be responsible for the observed decrease of aneuploid blastomeres from compaction to the peri-implantation stage.

The use of comparative genomic hybridization and fluorescent in situ hybridization in postmortem pathology investigation of congenital malformations (Article)

2010-03-01T00:00:00Z

Chromosomal abnormalities are an important cause of multiple congenital anomalies (MCA). However, conventional cytogenetic analysis using culture is unsuccessful in 10% to 40% of the cases. The purpose of this study was to examine if retrospective chromosomal analysis was possible on paraffin-embedded autopsy material with new techniques, including comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH). We investigated 92 patients, including 71 patients with MCA, 17 patients with an isolated congenital anomaly, and 4 normal controls, by conventional CGH analysis and/or FISH. The karyotype was known in 52 cases, of which 26 patients were normal and 26 had chromosomal anomalies. Comparative genomic hybridization or FISH confirmed all but 2 cases, which were not interpretable. In 40 patients the karyotype was unknown but could be analyzed successfully in 36 cases (90%) by CGH. In this series, we found 1 additional chromosomal aberration, 45,X (Turner syndrome). Furthermore, we examined the postmortem material of 12 patients by FISH, confirming a known abnormal karyotype in 9 patients, an abnormal karyotype found by CGH in 1 case, and confirming DiGeorge syndrome (22q11 deletion) in twins. Comparative genomic hybridization and FISH are reliable techniques with which to perform retrospective genetic analysis on paraffin-embedded autopsy material.

Interstitial 11q deletion derived from a maternal ins(4;11)(p14;q24.2q25): A patient report and review (Article)

2009-07-01T00:00:00Z

We present a family with multiple cytogenetic abnormalities, identified through a girl with several dysmorphic features and cardiac problems, suspected for Jacobsen syndrome. Cytogenetic analysis showed a 46,XX,del(11)(qter) karyotype, which was confirmed by fluorescence in situ hybridization (FISH). Cytogenetic investigation of the parents showed a chromosome aberration in both: the father had a t(11;12)(p13;q22) translocation and the mother was carrier of an ins(4;11)(p14;q24q25). FISH analysis with an 11q-subtelomeric probe from the secondgeneration telomere clone set and BACs from 11q24-q25 suggested a complex maternal rearrangement.However, subsequent array analysis showed a single interstitial deletion in the proband, derived from the maternal insertion. The aberrant karyotypes in both parents implicated an increased risk of unbalanced fetal chromosome composition, thus high risk for a child with multiple congenital abnormalities. Therefore, during the next pregnancy, the couple opted for prenatal diagnosis by means of amniocentesis. An interphase FISH strategy for uncultured amniotic fluid cells predicted two possible unbalanced fetal chromosome constitutions. Karyotyping of cultured amniotic cells confirmed one of the predicted unbalanced cytogenetic options, demonstrating the value of a fast interphase strategy for parents who both are carriers of a chromosomal abnormality. In addition, wepresent an overview of patients with Jacobsen syndrome and an interstitial 11q deletion reported thus far in literature.

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.

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

Does confined placental mosaicism account for adverse perinatal outcomes in IVF pregnancies? (Article)

2008-05-01T00:00:00Z

BACKGROUND: IVF singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. This may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. The aim of this study was to compare the incidence of confined placental mosaicism (CPM) in IVF/ICSI pregnancies and spontaneous conceptions. METHODS: We conducted a multi-centre retrospective analysis of karyotype results obtained by chorionic villus sampling (CVS), performed due to advanced maternal age (≥36 years at 18 weeks of gestation), in the Netherlands between 1995 and 2005. RESULTS: From a total of 322 246 pregnancies, 20 885 CVS results were analysed: 235 in the IVF/ICSI group and 20 650 in the control group. The mean age of women in both groups was 38.4 years (mean difference -0.08, 95% CI -0.35 to 0.18). Data relating to the fetal karyotype were missing in 143 cases in the control group. When taking into account missing data, the incidence of CPM was lower in the IVF-ICSI group than in the control group, 1.3% versus 2.2% (odds ratio 0.59, 95% CI 0.19-1.85), whereas the incidence of fetal chromosomal anomalies was increased 4.3% versus 2.4% (odds ratio 1.81, 95% CI 0.95-3.42). Neither differences were statistically significant. CONCLUSIONS: The incidence of CPM is not increased in IVF/ICSI pregnancies compared with spontaneous conceptions. CPM probably does not account for the adverse perinatal outcomes following IVF/ICSI.

Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: A randomized controlled trial (Article)

2007-04-01T00:00:00Z

BACKGROUND: To test whether ovarian stimulation for in-vitro fertilization (IVF) affects oocyte quality and thus chromosome segregation behaviour during meiosis and early embryo development, preimplantation genetic screening of embryos was employed in a prospective, randomized controlled trial, comparing two ovarian stimulation regimens. METHODS: Infertile patients under 38 years of age were randomly assigned to undergo a mild stimulation regimen using gonadotrophin-releasing hormone (GnRH) antagonist co-treatment (67 patients), which does not disrupt secondary follicle recruitment, or a conventional high-dose exogenous gonadotrophin regimen and GnRH agonist co-treatment (44 patients). Following IVF, embryos were biopsied at the eight-cell stage and the copy number of 10 chromosomes was analysed in 1 or 2 blastomeres. RESULTS: The study was terminated prematurely, after an unplanned interim analysis (which included 61% of the planned number of patients) found a lower embryo aneuploidy rate following mild stimulation. Compared with conventional stimulation, significantly fewer oocytes and embryos were obtained following mild stimulation (P < 0.01 and < 0.05, respectively). Consequently, both regimens generated on average a similar number (1.8) of chromosomally normal embryos. Differences in rates of mosaic embryos suggest an effect of ovarian stimulation on mitotic segregation errors. CONCLUSIONS: Future ovarian stimulation strategies should avoid maximizing oocyte yield, but aim at generating a sufficient number of chromosomally normal embryos by reduced interference with ovarian physiology.

A familial inverted duplication 2q33-q34 identified and delineated by multiple cytogenetic techniques (Article)

2007-03-01T00:00:00Z

We describe a unique family with two children having a delay in psychomotor development. In both children we identified an interstitial duplication dup(2)(q34q33) using multiple, complementary molecular cytogenetic techniques. Comparative genomic hybridisation (CGH) and array-CGH were used to determine the size and the location of the duplicated region, the orientation of the duplicated region was identified with fluorescence in situ hybridisation (FISH). Both parents demonstrated a normal karyotype and normal CGH and array-CGH-profiles. However, FISH on peripheral blood cells from the mother showed the inv dup(2) in 9% of metaphases and 19% of interphase nuclei. To our knowledge this is the first report of a mosaic carrier of duplication in the long arm of chromosome 2. The finding of chromosomal mosaicism of at least 19% in the mother increases the recurrence risk. The exact characterisation of the inv dup(2) with FISH probes enabled us to offer a reliable prenatal FISH test. Comparison of the clinical features of the two children with those of previously described cases supports the hypothesis that the characteristic facial phenotype is linked to the distal part of the 2q33-q37 region. This report illustrates that in case of two sibs with an identical structural chromosomal abnormality the possibility of parental chromosomal mosaicism must be thoroughly investigated.

FISH analysis of 15 chromosomes in human day 4 and 5 preimplantation embryos: The added value of extended aneuploidy detection (Article)

2007-01-01T00:00:00Z

Objective: Screening for an increased number of chromosomes may improve the detection of abnormal embryos and thus contribute to the capability of preimplantation genetic screening (PGS) to detect the embryo(s) for transfer in IVF with the best chance for a healthy child. Good-quality day 4 and 5 embryos were analyzed after cryopreservation for the nine chromosomes mostly recommended for screening (13, 14, 15, 16, 18, 21, 22, X and Y), next to six additional chromosomes which are less well studied in this context (1, 2, 7, 6, 10 and 17). Method: The copy numbers of 15 chromosomes were investigated by fluorescence in situ hybridization (FISH) in three consecutive rounds. The proportion of aneuploid and mosaic embryos was determined and compared in retrospect to results in case only the recommended probe set had been analyzed. Results: A total of 52 embryos from 29 infertile women were analyzed. Screening the embryos for six additional chromosomes increased the proportion of abnormal embryos from 67 to 81% (P = 0.03), owing to an increase in mosaic embryos. Conclusion: All but one of the meiotic aneuploidies found in this study would have been detected by the probe set most frequently used in PGS clinics. However, aneuploid cell lines originating from mitotic errors could be detected for almost all chromosomes, so screening of six additional chromosomes mainly increased the proportion of mosaic embryos. The added value of screening for six additional chromosomes in PGS for clinical practice will remain undetermined as long as the fate of mosaic embryos after transfer is unclear. Copyright