How genomics is changing the practice of prenatal testing.

New genomic laboratory technology namely microarrays and high throughput sequencing (HTS) as well as a steady progress in sonographic image capture and processing have changed the practice of prenatal diagnosis during the last decade fundamentally. Pregnancies at high risk for common trisomies are reliably identified by non-invasive prenatal testing (NIPT) and expert sonography has greatly improved the assessment of the fetal phenotype. Preconceptional comprehensive carrier screening using HTS is available for all parents, if they should wish to do so. A definite fetal diagnosis, however, will still require invasive testing for most conditions. Chromosomal microarrays (CMA) have greatly enhanced the resolution in the detection of chromosome anomalies and other causal copy number variations (CNV). Gene panel or whole exome sequencing (WES) is becoming the routine follow up of many anomalies detected by ultrasound after CNVs have been excluded. The benefits and limitations of the various screening as well as diagnostic options are perceived as complex by many who find it challenging to cope with the need for immediate choices. The communication of facts to ensure an informed decision making is obviously a growing challenge with the advent of the new genomic testing options. This contribution provides an overview of the current practice and policies in Switzerland.

Strømme Syndrome Is a Ciliary Disorder Caused by Mutations in CENPF.

Strømme syndrome was first described by Strømme et al. (1993) in siblings presenting with "apple peel" type intestinal atresia, ocular anomalies and microcephaly. The etiology remains unknown to date. We describe the long-term clinical follow-up data for the original pair of siblings as well as two previously unreported siblings with a severe phenotype overlapping that of the Strømme syndrome including fetal autopsy results. Using family-based whole-exome sequencing, we identified truncating mutations in the centrosome gene CENPF in the two nonconsanguineous Caucasian sibling pairs. Compound heterozygous inheritance was confirmed in both families. Recently, mutations in this gene were shown to cause a fetal lethal phenotype, the phenotype and functional data being compatible with a human ciliopathy [Waters et al., 2015]. We show for the first time that Strømme syndrome is an autosomal-recessive disease caused by mutations in CENPF that can result in a wide phenotypic spectrum.

Anterior segment dysgenesis associated with Williams-Beuren syndrome: a case report and review of the literature.

BACKGROUND: Williams-Beuren syndrome is characterized by mild mental retardation, specific neurocognitive profile, hypercalcemia during infancy, distinctive facial features and cardiovascular diseases. We report on complete ophthalmologic, sonographic and genetic evaluation of a girl with a clinical phenotype of Williams-Beuren syndrome, associated with unilateral anterior segment dysgenesis and bilateral cleft of the soft and hard palate. These phenotypic features have not been linked to the haploinsufficiency of genes involved in the microdeletion. CASE PRESENTATION: A term born girl presented at the initial examination with clouding of the right cornea. On ultrasound biomicroscopy the anterior chamber structures were difficult to differentiate, showing severe adhesions from the opacified cornea to the iris with a kerato-irido-lenticular contact to the remnant lens, a finding consistent with Peters' anomaly. Genetic analyses including FISH confirmed a loss of the critical region 7q11.23, usually associated with the typical Williams-Beuren syndrome. Microsatellite analysis showed a loss of about 2.36 Mb. CONCLUSIONS: A diagnosis of Williams-Beuren syndrome was made based on the microdeletion of 7q11.23. The unique features, including unilateral microphthalmia and anterior segment dysgenesis, were unlikely to be caused by the microdeletion. Arguments in favor of the latter are unilateral manifestation, as well as the fact that numerous patients with deletions of comparable or microscopically visible size have not shown similar manifestations.

mRNA transfection-based, feeder-free, induced pluripotent stem cells derived from adipose tissue of a 50-year-old patient.

Induced pluripotent stem cells (iPSC) have successfully been derived from somatic fibroblasts through transfection of synthetic modified mRNA encoding transcription factors. This technique obviates the use of recombinant DNA and viral vectors in cellular reprogramming. The present study derived iPSC from adipose-derived mesenchymal stem cells (of a 50-year-old female patient) by utilizing a similar technique, but with defined culture medium without feeder cells, during both reprogramming and propagation. Clonal selection was performed to yield 12 putative iPSC lines from individual colonies of nascent reprogrammed cells, starting from 150,000 cells. However, only seven lines maintained their undifferentiated state after 10 continuous serial passages. These seven lines were then subjected to a rigorous battery of analyses to confirm their identity as iPSC. These tests included immunostaining, flow cytometry, qRT-PCR, in vitro differentiation assay, and teratoma formation assay within SCID mice. Positive results were consistently observed in all analyses, thus verifying the cells as fully reprogrammed iPSC. While all 7 iPSC lines displayed normal karyogram up to passage 13, chromosomal anomalies occurred in 4 of 7 lines with extended in vitro culture beyond 24 serial passages. Only three lines retained normal karyotype of 46,XX. The remaining four lines displayed mosaicism of normal and abnormal karyotypes. Hence, this study successfully derived iPSC from abundant and easily accessible adipose tissues of a middle-aged patient; utilizing a mRNA-based integration-free technique under feeder-free conditions. This is a step forward in translating iPSC into personalized regenerative medicine within the clinic.

[Genetic testing in the fetus and child]. / Genetische Untersuchungen während der Schwangerschaft und beim Kind.

New key technologies such as array-based molecular karyotyping and high throughput sequencing are currently introduced in pre- and postnatal diagnostic testing. These greatly improved genomic testing approaches are beginning to fundamentally change diagnostic strategies in the clinical setting. Molecular karyotyping in the fetus is now routinely performed in high risk situations or on parental request. It will replace the conventional microscopic approach in the near future. Non-invasive prenatal testing to exclude common trisomies is probably the most significant recent achievement and has the potential to dramatically reduce invasive testing. Multiple congenital malformations and intellectual disability (ID) occur in up to 3 % of the general population. A correct diagnosis at an early age is important for clinical management of the patients and for counselling the families with regard to recurrence risk. Conventional karyotyping has been replaced by molecular karyotyping (microarray analysis, Array-CGH), increasing the diagnostic yield up to 15 - 20 % in this population. This approach can be challenging with regard to interpretation of copy number variants of uncertain significance or variants with reduced penetrance. If the clinical assessment leads to the suspicion of a specific syndrome or a leading symptom like epilepsy or microcephaly is present, genetic testing might be directed towards single-gene analysis. However, increasing knowledge indicates that many of these conditions are genetically heterogeneous. The availability of next-generation sequencing techniques has led to the implementation of testing panels in the diagnostic setting, by which multiple genes are analyzed in parallel. This approach allows for increased diagnostic yield in monogenic disorders and defining of more detailed genoptype-phenotype correlations. In addition, whole-exome or whole-genome sequencing has led to the identification of the genetic basis of many known genetic disorders and to the identification and delineation of novel disorders thus allowing a diagnosis in more patients. Fulfilling the potential of the increasing number of options for genetic testing for accurate diagnosis requires close collaboration between clinical geneticists and paediatricians.

Reduced expression by SETBP1 haploinsufficiency causes developmental and expressive language delay indicating a phenotype distinct from Schinzel-Giedion syndrome.

BACKGROUND: Mutations of the SET binding protein 1 gene (SETBP1) on 18q12.3 have recently been reported to cause Schinzel-Giedion syndrome (SGS). As rare 18q interstitial deletions affecting multiple genes including SETBP1 correlate with a milder phenotype, including minor physical anomalies and developmental and expressive speech delay, mutations in SETBP1 are thought to result in a gain-of-function or a dominant-negative effect. However, the consequence of the SETBP1 loss-of-function has not yet been well described. METHODS: Microarray-based comparative genomic hybridisation (aCGH) analyses were performed to identify genetic causes for developmental and expressive speech delay in two patients. SETBP1 expression in fibroblasts obtained from one of the patients was analysed by real-time RT-PCR and western blotting. A cohort study to identify nucleotide changes in SETBP1 was performed in 142 Japanese patients with developmental delay. RESULTS: aCGH analyses identified submicroscopic deletions of less than 1 Mb exclusively containing SETBP1. Both patients show global developmental, expressive language delay and minor facial anomalies. Decreased expression of SETBP1 was identified in the patient's skin fibroblasts. No pathogenic mutation of SETBP1 was identified in the cohort study. CONCLUSION: SETBP1 expression was reduced in a patient with SETBP1 haploinsufficiency, indicating that the SETBP1 deletion phenotype is allele dose sensitive. In correlation with the exclusive deletion of SETBP1, this study delimits a milder phenotype distinct from SGS overlapping with the previously described phenotype of del(18)(q12.2q21.1) syndrome including global developmental, expressive language delay and distinctive facial features. These findings support the hypothesis that mutations in SETBP1 causing SGS may have a gain-of-function or a dominant-negative effect, whereas haploinsufficiency or loss-of-function mutations in SETBP1 cause a milder phenotype.

Clinical, Cytogenetic and Molecular Cytogenetic Outcomes of Cell-Free DNA Testing for Rare Chromosomal Anomalies.

The scope of cell-free DNA (cfDNA) testing was expanded to the genome, which allowed screening for rare chromosome anomalies (RCAs). Since the efficiency of the test for RCAs remains below the common aneuploidies, there is a debate on the usage of expanded tests. This study focuses on the confirmatory and follow-up data of cases with positive cfDNA testing for RCAs and cases with screen-negative results in a series of 912 consecutive cases that underwent invasive testing following cfDNA testing. Chorion villus sampling (CVS), amniocentesis (AS), fetal blood sampling, and term placenta samples were investigated using classical cytogenetic and molecular cytogenetic techniques. Out of 593 screen-positive results, 504 (85%) were for common aneuploidies, 40 (6.7%) for rare autosomal trisomies (RATs), and 49 (8.3%) for structural chromosome anomalies (SAs). Of the screen-positives for RATs, 20 cases were evaluated only in fetal tissue, and confined placental mosaicism (CPM) could not be excluded. Among cases with definitive results (n = 20), the rates of true positives, placental mosaics, and false positives were 35%, 45%, and 10%, respectively. Among screen-positives for SAs, 32.7% were true positives. The confirmation rate was higher for duplications than deletions (58.3% vs. 29.4%). The rate of chromosomal abnormality was 10.9% in the group of 256 screen-negatives with pathological ultrasound findings. This study provides further data to assess the efficiency of expanded cfDNA testing for RATs and SAs. The test efficiency for cfDNA seems to be higher for duplications than for deletions, which is evidence of the role of expert ultrasound in identifying pregnancies at increased risk for chromosome anomalies, even in pregnancies with screen-negatives. Furthermore, we discussed the efficiency of CVS vs. AC in screen-positives for RATs.

aCGH on chorionic villi mirrors the complexity of fetoplacental mosaicism in prenatal diagnosis.

OBJECTIVE: To describe the diagnostic performance of array comparative genomic hybridization (aCGH) in the presence of mosaicism in the fetoplacental unit using direct chorionic villi. METHOD: In an ongoing study on the diagnostic performance of aCGH in 80 high-risk pregnancies, we studied three cases of placental mosaicism by carrying out aCGH on DNA of direct chorionic villi and chorionic villi cultures. RESULTS: Case 1: A three- to fourfold dosage gain of the region 18p in aCGH on direct villi was due to two additional isochromosomes 18p confined to the cytotrophoblast. Case 2: aCGH on direct villi revealed a normal result, whereas trisomy 18 mosaicism was present in cultured cells. Case 3: aCGH identifies monosomy X and mosaic disomy of the region Xp11.21-Xq12, whereas this mosaic cell line is not present in the conventional chromosome preparation on the cytotrophoblast. CONCLUSION: Although interpretation of aCGH results may be straightforward in the majority of cases, placental mosaicism may cause misinterpretations of rapid aCGH results on direct chorionic villi due to discrepant chromosomal constitutions of cytotrophoblast and mesenchymal villus core. Further investigations including cultures, fluorescence in situ hybridization and possible amniocentesis will still be required for interpretation of results.

Fetal polydactyly: a study of 24 cases ascertained by prenatal sonography.

Records of 24 pregnancies with fetal polydactyly were reviewed for the type of polydactyly, family history, associated sonographic findings, genetic testing, and postnatal/postmortem examination findings. The importance of fetal polydactyly can be mainly elucidated by the family history and absent or associated anomalies on a specialized malformation scan. Fetal karyotyping diagnoses frequent chromosomal anomalies in about half of cases with additional malformations, and array comparative genomic hybridization may be a future means of detecting cryptic chromosomal aberrations. Syndromic disorders of monogenic origin demand a careful interdisciplinary clinical assessment for establishing a clinical diagnosis and prognosis for the outcome of the child.

Interstitial deletion 1q42 in a patient with agenesis of corpus callosum: Phenotype-genotype comparison to the 1q41q42 microdeletion suggests a contiguous 1q4 syndrome.

Interstitial deletions of 1q4 are rare and present with different deletion breakpoints and variable phenotype. We report on the clinical and molecular cytogenetic findings in a girl with minor anomalies, midline defects including prenatally ascertained agenesis of the corpus callosum, epilepsy and developmental delay. A de novo 5.45 Mb deletion almost exclusively located within 1q42 was found to cause this phenotype, which shows significant overlap with the microdeletion 1q41q42 syndrome reported in a few patients except for the agenesis of the corpus callosum. However, deletions in patients with the 1q41q42 syndrome mainly extend into the 1q41 region with a region of overlap including the DISP1 gene involved in the SHH pathway, which is not part of the 1q42 deletion in our patient. We suggest that an interaction of genes involved in pathways of embryonic development rather than haploinsufficiency of single genes in the so-called critical regions is causing complex malformation syndromes due to cytogenetic microaberrations in the 1q4 region.

Expanding the spectrum of SMAD3-related phenotypes to agnathia-otocephaly.

BACKGROUND: Agnathia-otocephaly is a rare and lethal anomaly affecting craniofacial structures derived from the first pharyngeal arch. It is characterized by agnathia, microstomia, aglossia, and abnormally positioned auricles with or without associated anomalies. Variants affecting function of OTX2 and PRRX1, which together regulate the neural crest cells and the patterning of the first pharyngeal arch as well as skeletal and limb development, were identified to be causal for the anomaly in a few patients. METHODS: Family-based exome sequencing (ES) on a fetus with severe agnathia-otocephaly, cheilognathopalatoschisis, laryngeal hypoplasia, fused lung lobes and other organ abnormalities and mRNA expression analysis were performed. RESULTS: Exome sequencing detected a de novo SMAD3 missense variant in exon 6 (c.860G>A) associated with decreased mRNA expression. Variants in SMAD3 cause Loeys-Dietz syndrome 3 presenting with craniofacial anomalies such as mandibular hypoplasia, micro- or retro-gnathia, bifid uvula and cleft palate as well as skeletal anomalies and arterial tortuosity. The SMAD3 protein acts as a transcriptional regulator in the transforming growth factor ß (TGFB) and bone morphogenetic (BMP) signaling pathways, which play a key role in the development of craniofacial structures originating from the pharyngeal arches. CONCLUSION: Agnathia-otocephaly with or without associated anomalies may represent the severe end of a phenotypic spectrum related to variants in genes in the interacting SMAD/TGFB/BMP/SHH/FGF developmental pathways.

Analysis of severely affected patients with dihydropyrimidine dehydrogenase deficiency reveals large intragenic rearrangements of DPYD and a de novo interstitial deletion del(1)(p13.3p21.3).

Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14-16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3-1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.

Familial 14.5 Mb interstitial deletion 13q21.1-13q21.33: clinical and array-CGH study of a benign phenotype in a three-generation family.

We report on the clinical and cytogenetic findings as well as the array-based characterization of an interstitial familial 13q21 deletion initially recognized by standard karyotyping. Although 13q deletions are known to imply a wide variability of clinical consequences, the deletion carriers of the familial deletion in three generations did not reveal a relevant phenotype. The breakpoints and the deletion size in all three carrier individuals were determined by molecular karyotyping confirming a large 14.5 Mb deletion encompassing the 13q21.1-13q21.33 region identical in all three carriers. Gene paucity and the lack of dosage-sensitive genes in the delineated region might explain the apparently innocuous nature of this chromosomal anomaly. The example of this family presents evidence for describing the chromosomal region 13q21.1-13q21.33 as a large euchromatic variant or benign copy number variation without phenotypic consequences. Our data underline the importance of a phenogenetic approach combining clinical and laboratory evidence in the interpretation of segmental chromosomal anomalies especially in genetic counseling related to prenatal diagnosis.

Exome sequencing of fetal anomaly syndromes: novel phenotype-genotype discoveries.

The monogenic etiology of most severe fetal anomaly syndromes is poorly understood. Our objective was to use exome sequencing (ES) to increase our knowledge on causal variants and novel candidate genes associated with specific fetal phenotypes. We employed ES in a cohort of 19 families with one or more fetuses presenting with a distinctive anomaly pattern and/or phenotype recurrence at increased risk for lethal outcomes. Candidate variants were identified in 12 families (63%); in 6 of them a definite diagnosis was achieved including known or novel variants in recognized disease genes (MKS1, OTX2, FGFR2, and RYR1) and variants in novel disease genes describing new fetal phenotypes (CENPF, KIF14). We identified variants likely causal after clinical and functional review (SMAD3, KIF4A, and PIGW) and propose novel candidate genes (PTK7, DNHD1, and TTC28) for early human developmental disease supported by functional and cross-species phenotyping evidence. We describe rare and novel fetal anomaly syndromes and highlight the diagnostic utility of ES, but also its contribution to discovery. The diagnostic yield of the future application of prenatal ES will depend on our ability to increase our knowledge on the specific phenotype-genotype correlations during fetal development.

Rothmund-Thomson Syndrome: novel pathogenic mutations and frequencies of variants in the RECQL4 and USB1 (C16orf57) gene.

BACKGROUND: Poikiloderma is defined as a chronic skin condition presenting with a combination of punctate atrophy, areas of depigmentation, hyperpigmentation and telangiectasia. In a variety of hereditary syndromes such as Rothmund-Thomson syndrome (RTS), Clericuzio-type poikiloderma with neutropenia (PN) and Dyskeratosis Congenita (DC), poikiloderma occurs as one of the main symptoms. Here, we report on genotype and phenotype data of a cohort of 44 index patients with RTS or related genodermatoses. METHODS: DNA samples from 43 patients were screened for variants in the 21 exons of the RECQL4 gene using PCR, SSCP-PAGE analysis and/or Sanger sequencing. Patients with only one or no detectable mutation in the RECQL4 gene were additionally tested for variants in the 8 exons of the USB1 (C16orf57) gene by Sanger sequencing. The effect of novel variants was evaluated by phylogenic studies, single-nucleotide polymorphism (SNP) databases and in silico analyses. RESULTS: We identified 23 different RECQL4 mutations including 10 novel and one homozygous novel USB1 (C16orf57) mutation in a patient with PN. Moreover, we describe 31 RECQL4 and 8 USB1 sequence variants, four of them being novel intronic RECQL4 sequence changes that may have some deleterious effects on splicing mechanisms and need further evaluation by transcript analyses. CONCLUSION: The current study contributes to the improvement of genetic diagnostic strategies and interpretation in RTS and PN that is relevant in order to assess the patients' cancer risk, to avoid continuous and inconclusive clinical evaluations and to clarify the recurrence risk in the families. Additionally, it shows that the phenotype of more than 50% of the patients with suspected Rothmund-Thomson disease may be due to mutations in other genes raising the need for further extended genetic analyses.

Microdeletions in 9q33.3-q34.11 in five patients with intellectual disability, microcephaly, and seizures of incomplete penetrance: is STXBP1 not the only causative gene?

BACKGROUND: Most microdeletions involving chromosome sub-bands 9q33.3-9q34.11 to this point have been detected by analyses focused on STXBP1, a gene known to cause early infantile epileptic encephalopathy 4 and other seizure phenotypes. Loss-of-function mutations of STXBP1 have also been identified in some patients with intellectual disability without epilepsy. Consequently, STXBP1 is widely assumed to be the gene causing both seizures and intellectual disability in patients with 9q33.3-q34.11 microdeletions. RESULTS: We report five patients with overlapping microdeletions of chromosome 9q33.3-q34.11, four of them previously unreported. Their common clinical features include intellectual disability, psychomotor developmental delay with delayed or absent speech, muscular hypotonia, and strabismus. Microcephaly and short stature are each present in four of the patients. Two of the patients had seizures. De novo deletions range from 1.23 to 4.13 Mb, whereas the smallest deletion of 432 kb in patient 3 was inherited from her mother who is reported to have mild intellectual disability. The smallest region of overlap (SRO) of these deletions in 9q33.3 does not encompass STXBP1, but includes two genes that have not been previously associated with disease, RALGPS1 and GARNL3. Sequencing of the two SRO genes RALGPS1 and GARNL3 in at least 156 unrelated patients with mild to severe idiopathic intellectual disability detected no causative mutations. Gene expression analyses in our patients demonstrated significantly reduced expression levels of GARNL3, RALGPS1 and STXBP1 only in patients with deletions of the corresponding genes. Thus, reduced expression of STXBP1 was ruled out as a cause for seizures in our patient whose deletion did not encompass STXBP1. CONCLUSIONS: We suggest that microdeletions of this region on chromosome 9q cause a clinical spectrum including intellectual disability, developmental delay especially concerning speech, microcephaly, short stature, mild dysmorphisms, strabismus, and seizures of incomplete penetrance, and may constitute a new contiguous gene deletion syndrome which cannot completely be explained by deletion of STXBP1.

Gene expression profiles of similarly derived human embryonic stem cell lines correlate with their distinct propensity to exit stemness and their different differentiation behavior in culture.

Four normal-karyotype human embryonic stem cell (hESC) lines were generated using the same protocol and maintained under identical conditions. Despite these precautions, gene expression patterns were found to be dissimilar among the four lines. The observed differences were typical of each cell line, correlated with their distinct propensity to exit stemness, created heterogeneity among the cells during cell line maintenance, and correlated with their altered capacity as a source of differentiated cells. The capacity of some cell lines to give rise to more, and more mature, neurons within comparable time frames of directed differentiation reflected the distinct proportions of cells already predifferentiated at the onset. These findings demonstrate that the subsequent stages of neural differentiation were altered both in a quantitative and timely fashion. As a consequence, cell lines with apparent better and quicker ability to produce neurons were actually the less capable of reproducing proper differentiation. Previous data suggested that cell lines able to generate more neurons faster would be more suitable to clinical application. Our analysis of the differentiation process strongly suggests the opposite. The spontaneous tendency to predifferentiate of any particular hESC line should be known because it clearly impacts further experimental results.

Morphologic and GATA1 sequencing analysis of hematopoiesis in fetuses with trisomy 21.

Trisomy 21 alters fetal liver hematopoiesis and, in combination with somatic globin transcription factor 1 (GATA1) mutations, leads to development of transient myeloproliferative disease in newborns. However, little is known about the morphological hematopoietic changes caused by trisomy 21 in the fetus, and to date, the exact onset of GATA1 mutations remains uncertain. Therefore, we analyzed fetal liver hematopoiesis from second trimester pregnancies in trisomy 21 and screened for GATA1 mutations. We examined 57 formalin-fixed and paraffin-embedded fetal liver specimens (49 harboring trisomy 21 and 8 controls) by immunohistochemistry for CD34, CD61, factor VIII, and glycophorin A. GATA1 exon 2 was sequenced in fetal livers and corresponding nonhematologic tissue. Cell counts of megakaryocytes (P = .022), megakaryocytic precursors (P = .021), and erythroid precursors were higher in trisomy 21 cases. CD34-positive hematopoietic blasts showed no statistically significant differences. No mutation was detected by GATA1 exon 2 sequencing in fetal livers from 12 to 25 weeks of gestation. Our results suggest that GATA1 exon 2 mutations occur late in trisomy 21 fetal hematopoiesis. However, trisomy 21 alone provides a proliferative stimulus of fetal megakaryopoiesis and erythropoiesis. CD34-positive precursor cells are not increased in trisomy 21 fetal livers.