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We present a clinical case where a complex abnormal non-invasive prenatal test (NIPT) result in a research project revealed carcinoma of the breast in the pregnant woman. Furthermore, the NIPT result did not demonstrate the same fetal chromosomal aberration as the chorion villus sample. A literature search for similar cases was performed identifying 43 unique cases, where abnormal NIPT results were related to maternal malignancy. Malignancy is a rare but important cause of complex abnormal non-invasive prenatal test (NIPT) results and should be considered when fetal karyotype and abnormal NIPT results are discordant. Furthermore, a follow-up invasive sample is essential for correct fetal diagnosis when abnormal NIPT results are found.

INTRODUCTION Applying whole-exome sequencing (WES) for the diagnosis of diseases in children has shown significant diagnostic strength compared with chromosomal microarray. WES may also have the potential of adding clinically relevant prenatal information in cases where a fetus is found to have structural anomalies. We present results from the first fetal exomes performed in a tertiary center in Denmark. MATERIAL AND METHODS Couples/expectant parents were included in Central Denmark Region from July 2016 to March 2019. Inclusion was not systematic, but where one or more fetal malformations or severe fetal hydrops were detected, and a specific diagnosis had not been obtained by chromosomal microarray. WES was performed in ongoing pregnancies (N = 11), after intrauterine demise (N = 5), or after termination of pregnancy based on ultrasound findings (N = 19). In most cases, a trio format was applied comprising fetal and parental DNA. RESULTS WES was performed in 35 highly selected fetal cases. Pathogenic variants, or variants likely to explain the phenotype, were detected in 9/35 (26%). Variants of uncertain significance were detected in 7/35 (20%) and there was one secondary finding (3%). Out of the 11 ongoing pregnancies, four reached a genetic diagnosis (36%). Detection rate was highest in cases of multisystem anomalies (7/13, 54%). WES was completed in all three trimesters and both autosomal dominant, autosomal recessive and X-linked inheritance were revealed. CONCLUSIONS We present data from 35 cases of exome sequencing applied in a setting of fetal malformations. Importantly, though, we wish to share our personal experiences with implementing WES into a prenatal setting. As a medical society, we must continue to share what we do not understand, what went wrong, what is difficult, and what we do not agree upon. A common understanding and language are warranted. We also advocate that more research is needed concerning the clinical value, as well as costs and patient perspectives, of using WES in pregnancy. We believe that WES will lead to improved prenatal and perinatal care.

Background: The existing risk of procedure-related miscarriage following invasive sampling for prenatal diagnosis is higher for twin pregnancies and some women are reluctant to test these typically difficultly obtained pregnancies invasively. Therefore, there is a need for noninvasive testing options that can test twin pregnancies at an early gestational age and ideally test the twins individually.Case presentation: A pregnant woman opted for cell-based NIPT at GA 10 + 5. As cell-based NIPT is not established for use in twins, the test was provided in a research setting only, when an ultrasound scan showed that she carried dichorionic twins.Materials and Methods: Fifty mL of peripheral blood was sampled, and circulating fetal cells were enriched and isolated. Individual cells were subject to whole-genome amplification and STR analysis. Three fetal cells were analyzed by chromosomal microarray (aCGH).Results: We identified 20 fetal cells all sharing the same genetic profile, which increased the likelihood of monozygotic twins. aCGH of three fetal cells showed the presence of two X chromosomes and a gain of chromosome Y. CVS from both placentae confirmed the sex chromosomal anomaly, 47,XXY and that both fetuses were affected.Conclusion: NIPT options can provide valuable genetic information to twin pregnancies that help the couples in their decision-making on prenatal testing. Little has been published about the use of cell-based NIPT in twin pregnancies, but the method may offer the possibility to obtain individual cell-based NIPT results in dizygotic twins.

Pathogenic variants in the MED12 gene located on the X‐chromosome have primarily been reported in males with Lujan‐Fryns syndrome, Ohdo syndrome and the Opits‐Kaveggia syndrome. However, earlier reports of female patients and female mice suggest that MED12 deficiency causes severe malformations. We report a novel example of a MED12 de novo nonsense variant in a female fetus with severe malformations identified by trio‐exome sequencing. This finding further expands the clinical spectrum of MED12‐related disorders, which is vital for prenatal diagnosis and genetic counselling of couples., Pathogenic variants in MED12 located on the X‐chromosome are linked to intellectual disability and dysmorphism in males. We report a novel MED12 de novo nonsense variant in a female fetus with malformations, further expanding the clinical spectrum.

Noonan syndrome is characterized by variable phenotypic expressivity with characteristic dysmorphic facial features, varying degrees of intellectual disability, developmental delay, short stature, and congenital heart defects in 50–80%. Other findings include a webbed neck, cryptorchidism, coagulation defects and eye abnormalities.Thus far, Noonan syndrome has mainly been attributed to heterozygous pathogenic variants in 10+ different genes, with the rare exception of cases due to biallelic pathogenic variants in LZTR1. Recently, homozygous loss-of-function variants in SPRED2 have been identified as a cause of a recessive Noonan syndrome-like phenotype. We present the phenotypes of two additional patients with homozygosity for a previously unreported loss-of-function variant in SPRED2, thereby adding relevant clinical information about the recently described Noonan syndrome-like SPRED2-related phenotype.

Background: Cell-free NIPT and cell-based NIPT are risk-free testing options using maternal blood samples to screen for fetal aneuploidies, but the methods differ. For cell-free NIPT, the fetal fraction of cell-free DNA in plasma is analyzed with a high background of maternal DNA. In contrast, for cell-based NIPT, a limited number of the rare, intact fetal cells are isolated for the genetic analysis. This case demonstrates the differences regarding testing for fetal sex-chromosomes anomalies (SCAs) between these two tests.Materials and Methods: A pregnant woman with mosaicism for Turner syndrome opted for NIPT in first trimester. For the cell-free NIPT analysis, DNA extraction, genome-wide massive parallel sequencing, and data analysis were carried out as described by the kit manufacturer (Illumina©, San Diego, CA, USA). For cell-based NIPT, the first sample gave no result, but the woman consented to repeat cell-based NIPT. After whole genome amplification and STR analysis, fetal DNA from three individual fetal cells was subjected to chromosomal microarray (aCGH, Agilent oligoarray, 180 kb).Results: Fetal fraction was 7%, and cell-free NIPT showed 2 copies of chromosomes 13, 18, and 21 and a decreased proportion of chromosome X, suggestive of fetal Turner syndrome. In contrast, the cell-based NIPT result showed no aneuploidy and two X-chromosomes in the fetus.Conclusion: cell-based NIPT may provide a non-invasive testing option to screen for SCAs in women with mosaicism for monosomy-X in blood, where cell-free NIPT cannot discriminate whether the X-loss is maternal or fetal.

Xia-Gibbs syndrome (XGS) is a neurodevelopmental disorder characterized by intellectual disability, developmental delay, seizures, hypotonia, obstructive sleep apnoea and mild facial dysmorphism. Heterozygosity for loss-of-function variants in AHDC1, encoding the AT-hook DNA binding motif containing protein 1, were discovered in 2014 as the likely genetic cause of Xia-Gibbs syndrome. We present five patients with Xia-Gibbs syndrome caused by previously unreported variants in AHDC1. Two of the patients share a frameshift variant: c.2849del (p.(Pro950Argfs*192)) in AHDC1. Despite sharing this variant, the two patients show remarkable phenotypic differences underscoring the clinical heterogeneity of Xia-Gibbs syndrome. In addition, we present a case of Xia-Gibbs syndrome caused by mosaicism for an AHDC1 variant.

Aims: We aimed to reclassify a population-based cohort of 529 adult glioma patients to evaluate the prognostic impact of the 2016 World Health Organization (WHO) central nervous system tumour classification. Moreover, we evaluated the feasibility of gene panel next-generation sequencing (NGS) in daily diagnostics of 225 prospective glioma patients. Methods: The retrospective cohort was reclassified according to WHO 2016 criteria by immunohistochemistry for IDH-R132H, fluorescence in situ hybridization for 1p/19q-codeletion and gene panel NGS. All tumours of the prospective cohort were subjected to NGS analysis up-front. Results: The entire population-based cohort was successfully reclassified according to WHO 2016 criteria. NGS results were obtained for 98% of the prospective patients. Survival analyses in the population-based cohort confirmed three major prognostic subgroups, that is, isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas, IDH-mutant astrocytomas and IDH-wildtype glioblastomas. The distinction between WHO grade II and III was prognostic in patients with IDH-mutant astrocytoma. The survival of patients with IDH-wildtype diffuse astrocytomas carrying TERT promoter mutation and/or EGFR amplification overlapped with the poor survival of IDH-wildtype glioblastoma patients. Conclusions: Gene panel NGS proved feasible in daily diagnostics. In addition, our study confirms the prognostic role of glioma classification according to WHO 2016 in a large population-based cohort. Molecular features of glioblastoma in IDH-wildtype diffuse glioma were linked to poor survival corresponding to IDH-wildtype glioblastoma patients. The distinction between WHO grade II and III retained prognostic significance in patients with IDH-mutant diffuse astrocytic gliomas.

Assistive robotic arms (ARM) have the potential tomake individuals with tetraplegia more independent by enablingthem to perform physical activities. Usually the ARMs are usedwithin reach of the user while the user is sitting in a poweredwheelchair. In this proof of concept study, we demonstrated forthe first time a tongue based method to provide an individualwith tetraplegia the ability to fetch remote objects such asbeverages while being bedridden. This is done through tonguebasedcontrol of a mobile and remote assistive roboticmanipulator (ARM) which was mounted on a poweredwheelchair. The tongue-computer interface was used by twosubjects to remotely navigate an ARM carrying wheelchairtowards a table and control the ARM to pick up a bottle of waterplaced on the table. The subjects were given approximately 40min to become familiar with the system and then they performedthe task 10 times. Subject 1 succeeded the task in 9 out of 10 trialsand subject 2 in 8 out of 10 trials. The trials lasted for 132±38 sand 195±64 s for subject 1 and 2, respectively. Thus, the systemhas the potential to further empower individuals with tetraplegiaby enabling them to remotely pick-up objects when e.g. lying inbed, making them more independent and reducing the need forcare. Future work will include further development of thesystem (better feedback, semi automation) as well asexperimental assessment with more subjects and tasks.

Individuals suffering from quadriplegia can achieve increased independence by using an assistive robotic manipulator (ARM). However, due to their disability, the interfaces that can be used to operate such devices become limited. A versatile intraoral tongue control interface (ITCI) has previously been develop for this user group, as the tongue is usually spared from disability. A previous study has shown that the ITCI can provide direct and continuous control of 6-7 degrees of freedom (DoF) of an ARM, due to a high number of provided inputs (18). In the present pilot study we investigated whether semi-automation might further improve the efficiency of the ITCI, when controlling an ARM. This was achieved by adding a camera to the end effector of the ARM and using computer vision algorithms to guide the ARM to grasp a target object. Three ITCI and one joystick control scheme were tested and compared: 1) manual Cartesian control with a base frame reference point, 2) manual Cartesian control with an end effector reference point 3) manual Cartesian control with an end effector reference point and an autonomous grasp function 4) regular JACO2 joystick control. The results indicated that end effector control was superior to the base frame control in total task time, number of commands issued and path efficiency. The addition of the automatic grasp function did not improve the performance, but resulted in fewer collisions/displacements of the target object when grasping.

INTRODUCTION Targeted next-generation sequencing (NGS) offers promising diagnostic perspectives by making it possible to detect genetic alterations with high accuracy in multiple genes as part of the daily diagnostic work-up. The importance of genetic alterations is reflected in the 2016 WHO classification of CNS tumors, where specific alterations are incorporated in the definition of certain entities. The aim of this study was to assess the diagnostic potential of a customized targeted glioma-tailored 20-gene panel to identify molecular entities of gliomas and explore their distinct mutational profiles. Moreover, the prognostic impact of molecular diagnostics was explored. METHODS We used a setup with NGS, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) on a series of 756 glioma from both a retro- and prospective cohort. NGS data were obtained for 347 gliomas (225 analyzed prospectively, 122 analyzed retrospectively). RESULTS Three major clusters were identified by unsupervised hierarchial cluster analysis: 1) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytomas with frequent mutations in TP53 (83%) and ATRX (69%), 2) IDH-mutant and 1p/19q-codeleted oligodendrogliomas with frequent mutations in TERT (65%) and CIC (52%), and 3) IDH-wildtype astrocytic gliomas/glioblastomas with frequent mutations in TERT (32%), PTEN (28%) and TP53 (22%). Among the IHC IDH1-R132H negative gliomas, NGS detected other IDH mutations in 16% of the cases. Moreover, rare but diagnostically important mutations such as BRAF V600E and H3F3A K27M were detected. Survival analysis of the reclassified gliomas showed a clear difference in survival between patients with WHO 2007 and 2016 diagnoses. Successful prospective NGS analyses were obtained for 98% of the gliomas in daily diagnostics. CONCLUSION Application of NGS panel sequencing improves diagnostic accuracy and is feasible in daily diagnostics. NGS is ready to become part of standard diagnostics in the field of gliomas.

BACKGROUND: Lactase persistence is an autosomal dominant trait commonly distributed in Europe as well as some parts of east Africa and the Arabian Peninsula. Using real-time PCR to detect the -13910C > T variant common in the European population is a reliable analysis although other variants in the probe-binding site may cause errors in analysis. The aim of this study was to determine the prevalence of the variants in a Danish cohort examined for lactose intolerance as well as to improve the real-time PCR analysis for detection of the different variants.METHODS: We genotyped 3395 routine samples using real-time PCR for the -13910C > T-variant. All consecutive samples identified as -13910CC were sequenced using Sanger Sequencing. Using the SDS software we examined various quality value settings to improve on the genetic analysis.RESULTS: Using real-time PCR resulted in 100% successful genotyping of the -13910C > T variant. By using a quality value of 99% and sequencing the undetermined samples we improved the ability of the assay to identify variants other than -13910C > T. This resulted in a reduction of the diagnostic error rate by a factor of 2.4 while increasing the expenses only 3%.CONCLUSIONS: We conclude that using a quality value of 99% in the SDS software significantly improves the diagnostic efficiency of the real-time PCR assay for detecting variants associated to lactase persistence.

Hydatidiform mole is an abnormal human pregnancy characterized by the fetus being absent or nonviable, and the chorionic villi being vesicular and with trophoblastic hyperplasia. Most often, the mole phenotype is seen in conceptuses with an excess of paternally inherited genome set(s) relative to maternally inherited genome set(s), suggesting that the phenotype is caused by an excess of genome with a paternal imprinting pattern. However, it is unknown if correct parental origin of every imprinted gene is crucial for normal early differentiation or if abnormal parental imprinting of only one, or some, gene(s) can cause the mole phenotype. Two conceptuses included in the Danish Mole Project stood out since they presented with vesicular chorionic villi and without signs of fetal differentiation, and had apparently biparental diploid genomes, and no mutations in NLRP7 or KHDC3L were detected in the mothers. These conceptuses were subjected to a centralized histopathological revision and their genetic complements were scrutinized using fluorescence in situ hybridization, and DNA-marker and array comparative genomic hybridization analyses. Both conceptuses showed dysmorphic chorionic villi with some similarities to hydatidiform moles; however, no definite florid trophoblast hyperplasia was observed. Both conceptuses showed paternal hemizygosity of 11pter-11p15.4, most likely in nonmosaic state. Our findings suggest that the product of one (or a few) maternally expressed gene(s) on the tip of chromosome 11 is necessary for normal early embryonic differentiation. However, since the present two cases did not exhibit all features of hydatidiform moles, it is likely that abnormal parental imprinting of genes in other regions contribute to the phenotype of a hydatidiform mole.

To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation., Author Summary Gene expression is controlled at many different levels to assure appropriate responses to internal and environmental changes. The effect of topological changes in the DNA double helix on gene transcription in vivo is a poorly understood factor in the regulation of eukaryotic gene expression. Topological changes are constantly generated by DNA tracking processes and may influence gene expression if not constantly removed by DNA topoisomerases. For decades it has been generally accepted that these enzymes regulate transcription by removing excess topological strain generated during tracking of the RNA polymerase, but we still lack a more holistic view of how these enzymes influence gene transcription in their native environment. Here, we examine both global and gene-specific changes in transcription following lack of DNA topoisomerases in budding yeast. Taken together, our findings show that topoisomerases play a profound role during transcriptional activation of genes with a repressible/inducible mode of regulation. For the PHO5 gene, which is investigated in more detail, we demonstrate that topoisomerases are required for binding of a transcription factor, which is crucial for promoter opening during PHO5 activation. Our data thus suggest that inducible gene promoters are highly sensitive to changes in DNA superhelicity.