Clinical impact of additional findings detected by genome-wide non-invasive prenatal testing: Follow-up results of the TRIDENT-2 study.

In the TRIDENT-2 study, all pregnant women in the Netherlands are offered genome-wide non-invasive prenatal testing (GW-NIPT) with a choice of receiving either full screening or screening solely for common trisomies. Previous data showed that GW-NIPT can reliably detect common trisomies in the general obstetric population and that this test can also detect other chromosomal abnormalities (additional findings). However, evidence regarding the clinical impact of screening for additional findings is lacking. Therefore, we present follow-up results of the TRIDENT-2 study to determine this clinical impact based on the laboratory and perinatal outcomes of cases with additional findings. Between April 2017 and April 2019, additional findings were detected in 402/110,739 pregnancies (0.36%). For 358 cases, the origin was proven to be either fetal (n = 79; 22.1%), (assumed) confined placental mosaicism (CPM) (n = 189; 52.8%), or maternal (n = 90; 25.1%). For the remaining 44 (10.9%), the origin of the aberration could not be determined. Most fetal chromosomal aberrations were pathogenic and associated with severe clinical phenotypes (61/79; 77.2%). For CPM cases, occurrence of pre-eclampsia (8.5% [16/189] vs 0.5% [754/159,924]; RR 18.5), and birth weight <2.3rd percentile (13.6% [24/177] vs 2.5% [3,892/155,491]; RR 5.5) were significantly increased compared to the general obstetric population. Of the 90 maternal findings, 12 (13.3%) were malignancies and 32 (35.6%) (mosaic) pathogenic copy number variants, mostly associated with mild or no clinical phenotypes. Data from this large cohort study provide crucial information for deciding if and how to implement GW-NIPT in screening programs. Additionally, these data can inform the challenging interpretation, counseling, and follow-up of additional findings.

Prenatal ultrasound findings of rasopathies in a cohort of 424 fetuses: update on genetic testing in the NGS era.

BACKGROUND: This study evaluates 6 years of prenatal rasopathy testing in the Netherlands, updates on previous data and gives recommendations for prenatal rasopathy testing. METHODS: 424 fetal samples, sent in for prenatal rasopathy testing in 2011-2016, were collected. Cohort 1 included 231 samples that were sequenced for 1-5 rasopathy genes. Cohort 2 included 193 samples that were analysed with a 14-gene next generation sequencing (NGS) panel. For all mutation-positive samples in both cohorts, the referring physician provided detailed ultrasound findings and postnatal follow-up. For 168 mutation-negative samples in cohort 2, solely clinical information on the requisition form was collected. RESULTS: In total, 40 (likely) pathogenic variants were detected (9.4%). All fetuses showed a variable degree of involvement of prenatal findings: increased nuchal translucency (NT)/cystic hygroma, distended jugular lymph sacs (JLS), hydrops fetalis, polyhydramnios, pleural effusion, ascites, cardiac defects and renal anomalies. An increased NT was the most common finding. Eight fetuses showed solely an increased NT/cystic hygroma, which were all larger than 5.5 mm. Ascites and renal anomalies appeared to be poor predictors of pathogenic outcome. CONCLUSION: Fetuses with a rasopathy show in general multiple ultrasound findings. The larger the NT and the longer it persists, the more likely it is to find a pathogenic variant. Rasopathy testing is recommended when the fetus shows an isolated increased NT ≥5.0 mm or when NT of ≥3.5 mm and at least one of the following ultrasound anomalies is present: distended JLS, hydrops fetalis, polyhydramnios, pleural effusion, ascites, cardiac defects and renal anomalies.

Deficiency of the human cysteine protease inhibitor cystatin M/E causes hypotrichosis and dry skin.

PURPOSE: We aimed to assess the biological and clinical significance of the human cysteine protease inhibitor cystatin M/E, encoded by the CTS6 gene, in diseases of human hair and skin. METHODS: Exome and Sanger sequencing was performed to reveal the genetic cause in two related patients with hypotrichosis. Immunohistochemical, biophysical, and biochemical measurements were performed on patient skin and 3D-reconstructed skin from patient-derived keratinocytes. RESULTS: We identified a homozygous variant c.361C>T (p.Gln121*), resulting in a premature stop codon in exon 2 of CST6 associated with hypotrichosis, eczema, blepharitis, photophobia and impaired sweating. Enzyme assays using recombinant mutant cystatin M/E protein, generated by site-directed mutagenesis, revealed that this p.Gln121* variant was unable to inhibit any of its three target proteases (legumain and cathepsins L and V). Three-dimensional protein structure prediction confirmed the disturbance of the protease/inhibitor binding sites of legumain and cathepsins L and V in the p.Gln121* variant. CONCLUSION: The herein characterized autosomal recessive hypotrichosis syndrome indicates an important role of human cystatin M/E in epidermal homeostasis and hair follicle morphogenesis.

Prenatal diagnosis of cervical ribs by three-dimensional ultrasound in a foetus with a herniated Dandy-Walker cyst.

We present a case report of a foetus with a herniated Dandy-Walker cyst and bilateral rudimentary cervical ribs. The cervical ribs were visualised prenatally by three-dimensional ultrasound and confirmed by post-termination radiography. The prevalence of cervical ribs is higher in deceased fetuses and neonates with or without structural abnormalities compared with healthy individuals and might be regarded as a marker of disadvantageous fetal development. We demonstrate that evaluation of the fetal vertebral pattern by three-dimensional ultrasonography, including the cervical region, is feasible and could provide valuable information regarding fetal and neonatal prognosis.

Pitfalls in the diagnosis of hemophilia severity: What to do?

Measurements of factor VIII coagulation activity (FVIII:C) may vary and result in misclassification of hemophilia A with delay in initiation of prophylactic treatment. We describe two young brothers who were diagnosed as moderate hemophilia patients and therefore not prophylactically treated with factor VIII concentrate despite frequent bleeding events. These findings emphasize the importance of (i) multiple measurements of FVIII:C by certified laboratories, (ii) adjustment of treatment when test results do not correspond to clinical symptoms, (iii) relevance of additional DNA mutation analysis in patients with hemophilia A, and (iv) treatment in centers with expertise.

Open-access flexible sigmoidoscopy frequently leads to additional colonoscopy in symptomatic patients over 50 years.

BACKGROUND & AIMS: General practitioners (GPs) in the Netherlands have open access to flexible sigmoidoscopy (FS) for patients with lower gastrointestinal symptoms, but not to colonoscopy. This study was performed to investigate the yield of FS in GP-referred patients, to evaluate the proportion of patients in whom additional colonoscopy was performed and to investigate whether there was a subgroup of patients referred for symptoms with a low risk of detecting significant findings. METHODS: All patients undergoing FS in 2008 and 2009 who were referred by GPs were analyzed. Indications for additional colonoscopy were the presence of polyps and/or colorectal cancer (CRC), polyp screening or surveillance, incomplete FS or other reasons. RESULTS: In total, 916 patients underwent FS. A cause for the symptoms was found in 44.2% of patients. In patients aged 50 years or older, additional colonoscopy was more frequently performed than in younger patients (27.5% vs. 9.6%, OR=3.6 [95% CI 2.4-5.4]), mainly due to a higher prevalence of adenomatous polyps (29.9% vs. 10.5%, OR=3.6 [95% CI 2.4-5.4]) and CRC (7.5% vs. 1.3%, OR=6.2 [95% CI 2.2-17.5]) during FS. In 7.8% patients undergoing FS for abdominal pain as the presenting symptom, a probable cause for the symptoms was found, mainly diverticular disease. CONCLUSION: Due to the high prevalence of polyps and CRC in symptomatic patients aged 50 years or older undergoing FS, an additional colonoscopy is performed frequently. In patients referred with abdominal pain, FS is unlikely to reveal a relevant cause for the symptoms.

Application of SNP array for rapid prenatal diagnosis: implementation, genetic counselling and diagnostic flow.

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.

Translational control of putative protooncogene Nm23-M2 by cytokines via phosphoinositide 3-kinase signaling.

The expansion and differentiation of hematopoietic progenitors is regulated by cytokine and growth factor signaling. To examine how signal transduction controls the gene expression program required for progenitor expansion, we screened ATLAS filters with polysome-associated mRNA derived from erythroid progenitors stimulated with erythropoietin and/or stem cell factor. The putative proto-oncogene nucleoside diphosphate kinase B (ndpk-B or nm23-M2) was identified as an erythropoietin and stem cell factor target gene. Factor-induced expression of nm23-M2 was regulated specifically at the level of polysome association by a phosphoinositide 3-kinase-dependent mechanism. Identification of the transcription initiation site revealed that nm23-M2 mRNA starts with a terminal oligopyrimidine sequence, which is known to render mRNA translation dependent on mitogenic factors. Recently, the nm23-M2 locus was identified as a common leukemia retrovirus integration site, suggesting that it plays a role in leukemia development. The expression of Nm23 from a retroviral vector in the absence of its 5'-untranslated region caused constitutive polysome association of nm23-M2. Polysome-association and protein expression of endogenous nm23-M2 declined during differentiation of erythroid progenitors, suggesting a role for Nm23-M2 in progenitor expansion. Taken together, nm23-m2 exemplifies that cytokine-dependent control of translation initiation is an important mechanism of gene expression regulation.

Large-scale identification of novel potential disease loci in mouse leukemia applying an improved strategy for cloning common virus integration sites.

The identification of common virus integration sites (cVIS) in retrovirally induced tumors in mice provides a powerful strategy to isolate novel transforming genes. Applying virus LTR-specific inverse-PCR and RT-PCR combined with automated sequencing on CasBr-M Murine Leukemia Virus (MuLV) induced myeloid leukemias, 126 virus integration sites were cloned. Using locus- and LTR-specific primers, a nested-PCR/Southern-blotting procedure was developed on genomic DNA from a large panel of MuLV-induced leukemias, to analyse whether a particular virus insertion represented a cVIS. In fact 39 out of 41 integrations analysed this way appeared to represent a common virus integration. We recognized six previously cloned cVISs, i.e. Evi1, Hoxa7, c-Myb, Cb2/Evi11, Evi12, and His1 and 33 novel common insertions, designated Cas-Br Virus Integration Site (Casvis). Among this group we found integrations in or near genes encoding nuclear proteins, e.g. Dnmt-2, Nm23-M2, Ctbp1 or Erg, within receptor genes, e.g. Cb2 or mrc1, novel putative signaling or transporter genes, the ringfinger-protein gene Mid1 and a panel of genes encoding novel proteins with unknown function. The finding that 39 out of 41 integrations analysed represented a cVIS, suggests that the majority of the other virus insertions that were not yet analysed by the PCR/Southern-blotting method are located in a cVIS as well and may therefore also harbor novel disease genes.

Leukemic predisposition of pSca-1/Cb2 transgenic mice.

OBJECTIVE: The gene encoding the peripheral cannabinoid receptor Cb2 is located in the common virus integration site Evi11 and is associated with hematopoietic malignancies in mice. To determine the effect of Cb2 overexpression on hematopoietic development in vivo, Cb2 transgenic mice were generated. MATERIALS AND METHODS: A Cb2 expression vector was constructed containing a Cb2 cDNA fragment cloned into the 14kb Sca-1 (Ly-6E.1) gene. Two transgenic lines in which Cb2 expression is controlled by the Sca-1 promoter were generated, and the effect on hematopoietic development was studied. Expression of Cb2 mRNA or protein was studied by RNase protection analysis and ligand binding assays, respectively. Leukemic predisposition was investigated by injecting newborn transgenic as well as control animals with Cas-Br-M murine leukemia virus (Cas-Br-M MuLV). RESULTS: Although increased expression of the Cb2 gene was observed in hematopoietic tissues, follow-up of more than 1 year did not reveal any hematologic defect. Interestingly, infection of newborn pSca-1/Cb2 transgenic mice with Cas-Br-M MuLV revealed that significantly more transgenic mice developed leukemia than virus-treated control littermates. Because these studies provide evidence for the cooperative potential of Cb2 in leukemia progression, we wished to identify genes that may collaborate with Cb2 in leukemic transformation. Our study suggests that Evi1, another common target for proviral integration in mouse leukemias, may be overexpressed in virus-induced leukemias in pSca-1/Cb2 transgenic mice. CONCLUSIONS: The data indicate that hematopoietic precursor cells that express high levels of Cb2 possess increased susceptibility for leukemia development and that Cb2 and Evi1 might collaborate in leukemogenesis.