Publications by authors named "Eric Legius"

151 Publications

Challenges in Treating Genodermatoses: New Therapies at the Horizon.

Front Pharmacol 2021 5;12:746664. Epub 2022 Jan 5.

Department of Pediatric Dermatology and Dermatology, National Reference Centre for Genodermatosis and Rare Diseases of the Skin (MAGEC), Hôpital Necker-Enfants Malades, and Assistance Publique-Hôpitaux de Paris, Université Paris Descartes, ERN Skin, Paris, France.

Genodermatoses are rare inherited skin diseases that frequently affect other organs. They often have marked effects on wellbeing and may cause early death. Progress in molecular genetics and translational research has unravelled many underlying pathological mechanisms, and in several disorders with high unmet need, has opened the way for the introduction of innovative treatments. One approach is to intervene where cell-signaling pathways are dysregulated, in the case of overactive pathways by the use of selective inhibitors, or when the activity of an essential factor is decreased by augmenting a molecular component to correct disequilibrium in the pathway. Where inflammatory reactions have been induced by a genetically altered protein, another possible approach is to suppress the inflammation directly. Depending on the nature of the genodermatosis, the implicated protein or even on the particular mutation, to correct the consequences or the genetic defect, may require a highly personalised stratagem. Repurposed drugs, can be used to bring about a "read through" strategy especially where the genetic defect induces premature termination codons. Sometimes the defective protein can be replaced by a normal functioning one. Cell therapies with allogeneic normal keratinocytes or fibroblasts may restore the integrity of diseased skin and allogeneic bone marrow or mesenchymal cells may additionally rescue other affected organs. Genetic engineering is expanding rapidly. The insertion of a normal functioning gene into cells of the recipient is since long explored. More recently, genome editing, allows reframing, insertion or deletion of exons or disruption of aberrantly functioning genes. There are now several examples where these stratagems are being explored in the (pre)clinical phase of therapeutic trial programmes. Another stratagem, designed to reduce the severity of a given disease involves the use of RNAi to attenuate expression of a harmful protein by decreasing abundance of the cognate transcript. Most of these strategies are short-lasting and will thus require intermittent life-long administration. In contrast, insertion of healthy copies of the relevant gene or editing the disease locus in the genome to correct harmful mutations in stem cells is more likely to induce a permanent cure. Here we discuss the potential advantages and drawbacks of applying these technologies in patients with these genetic conditions. Given the severity of many genodermatoses, prevention of transmission to future generations remains an important goal including offering reproductive choices, such as preimplantation genetic testing, which can allow selection of an unaffected embryo for transfer to the uterus.
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http://dx.doi.org/10.3389/fphar.2021.746664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8766835PMC
January 2022

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Am J Hum Genet 2021 09 26;108(9):1669-1691. Epub 2021 Jul 26.

Phoenix Children's Hospital, Phoenix, AZ 85016, USA; Department of Child Health, University of Arizona College of Medicine Phoenix, Phoenix, AZ 85004, USA.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8456166PMC
September 2021

MEK inhibition ameliorates social behavior phenotypes in a Spred1 knockout mouse model for RASopathy disorders.

Mol Autism 2021 07 26;12(1):53. Epub 2021 Jul 26.

Department of Human Genetics, KU Leuven, O&N1 Herestraat 49, Box 607, 3000, Leuven, Belgium.

Background: RASopathies are a group of disorders that result from mutations in genes coding for proteins involved in regulating the Ras-MAPK signaling pathway, and have an increased incidence of autism spectrum disorder (ASD). Legius syndrome is a rare RASopathy caused by loss-of-function mutations in the SPRED1 gene. The patient phenotype is similar to, but milder than, Neurofibromatosis type 1-another RASopathy caused by loss-of-function mutations in the NF1 gene. RASopathies exhibit increased activation of Ras-MAPK signaling and commonly manifest with cognitive impairments and ASD. Here, we investigated if a Spred1-/- mouse model for Legius syndrome recapitulates ASD-like symptoms, and whether targeting the Ras-MAPK pathway has therapeutic potential in this RASopathy mouse model.

Methods: We investigated social and communicative behaviors in Spred1-/- mice and probed therapeutic mechanisms underlying the observed behavioral phenotypes by pharmacological targeting of the Ras-MAPK pathway with the MEK inhibitor PD325901.

Results: Spred1-/- mice have robust increases in social dominance in the automated tube test and reduced adult ultrasonic vocalizations during social communication. Neonatal ultrasonic vocalization was also altered, with significant differences in spectral properties. Spred1-/- mice also exhibit impaired nesting behavior. Acute MEK inhibitor treatment in adulthood with PD325901 reversed the enhanced social dominance in Spred1-/- mice to normal levels, and improved nesting behavior in adult Spred1-/- mice.

Limitations: This study used an acute treatment protocol to administer the drug. It is not known what the effects of longer-term treatment would be on behavior. Further studies titrating the lowest dose of this drug that is required to alter Spred1-/- social behavior are still required. Finally, our findings are in a homozygous mouse model, whereas patients carry heterozygous mutations. These factors should be considered before any translational conclusions are drawn.

Conclusions: These results demonstrate for the first time that social behavior phenotypes in a mouse model for RASopathies (Spred1-/-) can be acutely reversed. This highlights a key role for Ras-MAPK dysregulation in mediating social behavior phenotypes in mouse models for ASD, suggesting that proper regulation of Ras-MAPK signaling is important for social behavior.
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http://dx.doi.org/10.1186/s13229-021-00458-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8314535PMC
July 2021

Lessons learned from drug trials in neurofibromatosis: A systematic review.

Eur J Med Genet 2021 Sep 5;64(9):104281. Epub 2021 Jul 5.

Department of General Paediatrics, Sophia's Children's Hospital, Rotterdam, the Netherlands; ENCORE, Erasmus MC Rotterdam, the Netherlands; Full Member of the European Reference Network on Genetic Tumour Risk Syndromes, (ERN GENTURIS)-Project ID No 739547, UK. Electronic address:

Neurofibromatosis (NF) is the umbrella term for neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2) and schwannomatosis (SWN). EU-PEARL aims to create a framework for platform trials in NF. The aim of this systematic review is to create an overview of recent clinical drug trials in NF, to identify learning points to guide development of the framework. We searched Embase, Medline and Cochrane register of trials on October 1, 2020 for publications of clinical drug trials in NF patients. We excluded publications published before 2010, systematic reviews, secondary analyses and studies with <10 patients. Data was extracted on manifestations studied, study design, phase, number of participating centres and population size. Full-text review resulted in 42 articles: 31 for NF1, 11 for NF2, none for SWN. Most NF1 trials focused on plexiform neurofibromas (32%). Trials in NF2 solely studied vestibular schwannomas. In NF1, single-arm trials (58%) were most common, and the majority was phase II (74%). For NF2 most trials were single-arm (55%) and exclusively phase II. For both diseases, trials were predominantly single-country and included five centres or less. Study population sizes were small, with the majority including ≤50 patients (74%). In conclusion, NF research is dominated by studies on a limited number out of the wide range of manifestations. We need more trials for cutaneous manifestations and high-grade gliomas in NF1, manifestations other than vestibular schwannoma in NF2 and trials for SWN. Drug development in NF may profit from innovative trials on multiple interventions and increased international collaboration.
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http://dx.doi.org/10.1016/j.ejmg.2021.104281DOI Listing
September 2021

Comprehensive genome-wide analysis of routine non-invasive test data allows cancer prediction: A single-center retrospective analysis of over 85,000 pregnancies.

EClinicalMedicine 2021 May 13;35:100856. Epub 2021 May 13.

Department of Oncology, KU Leuven, Herestraat 49, Leuven, Belgium.

Background: Implausible false positive results in non-invasive prenatal testing (NIPT) have been occasionally associated with the detection of occult maternal malignancies. Hence, there is a need for approaches allowing accurate prediction of whether the NIPT result is pointing to an underlying malignancy, as well as for organized programs ensuring efficient downstream clinical management of these cases.

Methods: Using a data set of 88,294 NIPT performed at University Hospital Leuven (Belgium) between November 2013 and March 2020, we retrospectively evaluated the positive predictive value (PPV) of our NIPT approach for cancer detection. In this approach, whole-genome cell-free DNA (cfDNA) data from NIPT were scrutinized for the presence of (sub)chromosomal copy number alterations (CNAs) predictive for a malignancy, using an unbiased NIPT analysis pipeline coined GIPSeq. For suspected cases, the presence of a maternal cancer was evaluated via subsequent multidisciplinary clinical follow-up examinations. The cancer-specificity of the identified CNAs in cfDNA was assessed through genetic analyses of a tumor biopsy.

Findings: Fifteen women without a cancer history were identified with a GIPSeq result suggestive of a malignant process. Their cfDNA profiles showed either genome-wide aberrations or a single trisomy 8. Upon clinical examinations, a solid or hematological cancer was identified in 4 and 7 cases, respectively. Three women were identified as having a clonal mosaicism. For one case no underlying condition was found. These numbers add to a PPV of 73%. Based on this experience, we presented a multidisciplinary care path for efficient clinical management of these cases.

Interpretation: The presented approach for analysing NIPT results has a high PPV, yet unknown sensitivity, for detecting asymptomatic malignancies upon routine NIPT. Given the complexity of diagnosing a pregnant woman with cancer, clinical follow-up should occur in a well-designed multidisciplinary setting, such as via the care model that we presented here.

Funding: This work was supported by Research Foundation Flanders and KU Leuven funding.
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http://dx.doi.org/10.1016/j.eclinm.2021.100856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138727PMC
May 2021

Revised diagnostic criteria for neurofibromatosis type 1 and Legius syndrome: an international consensus recommendation.

Genet Med 2021 08 19;23(8):1506-1513. Epub 2021 May 19.

Department of Genomic Medicine, St Mary's Hospital, Manchester Academic Health Sciences Centre (MAHSC), Division of Evolution and Genomic Science, University of Manchester, Manchester, UK.

Purpose: By incorporating major developments in genetics, ophthalmology, dermatology, and neuroimaging, to revise the diagnostic criteria for neurofibromatosis type 1 (NF1) and to establish diagnostic criteria for Legius syndrome (LGSS).

Methods: We used a multistep process, beginning with a Delphi method involving global experts and subsequently involving non-NF experts, patients, and foundations/patient advocacy groups.

Results: We reached consensus on the minimal clinical and genetic criteria for diagnosing and differentiating NF1 and LGSS, which have phenotypic overlap in young patients with pigmentary findings. Criteria for the mosaic forms of these conditions are also recommended.

Conclusion: The revised criteria for NF1 incorporate new clinical features and genetic testing, whereas the criteria for LGSS were created to differentiate the two conditions. It is likely that continued refinement of these new criteria will be necessary as investigators (1) study the diagnostic properties of the revised criteria, (2) reconsider criteria not included in this process, and (3) identify new clinical and other features of these conditions. For this reason, we propose an initiative to update periodically the diagnostic criteria for NF1 and LGSS.
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http://dx.doi.org/10.1038/s41436-021-01170-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8354850PMC
August 2021

Identifying challenges in neurofibromatosis: a modified Delphi procedure.

Eur J Hum Genet 2021 Nov 26;29(11):1625-1633. Epub 2021 Apr 26.

Department of General Paediatrics, Sophia's Children's Hospital, Rotterdam, The Netherlands.

Neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2) and schwannomatosis (SWN) are rare conditions with pronounced variability of clinical expression. We aimed to reach consensus on the most important manifestations meriting the development of drug trials. The five-staged modified Delphi procedure consisted of two questionnaires and a consensus meeting for 40 NF experts, a survey for 63 patient representatives, and a final workshop. In the questionnaires, manifestations were scored on multiple items on a 4-point Likert scale. The highest average scores for NF experts deciding the 'need for new treatment' were for malignant peripheral nerve sheath tumour (MPNST) (4,0) and high grade glioma (HGG) (3,9) for NF1; meningioma (3,9) for NF2 and pain (3,9) for SWN. The patient representatives assigned high scores to all manifestations, with plexiform neurofibroma being highest in NF1 (4,0), vestibular schwannoma in NF2 (4,0), and pain in SWN (3,9). Twelve experts participated in the consensus meeting and prioritised manifestations. MPNST was ranked the highest for NF1, followed by benign peripheral nerve sheath tumours. Tumour manifestations received highest ranking in NF2, and pain was the most prominent problem for SWN. Patient representative ratings for NF1 were similar to the experts' opinions, except that they ranked HGG as the most important manifestation. For NF2 and SWN, the patient representatives agreed with the experts. We conclude that NF experts and patient representatives consent to prioritise development of drug trials for MPNST, benign peripheral nerve sheath tumours, cutaneous manifestations and HGG for NF1; tumours for NF2; and pain for SWN.
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http://dx.doi.org/10.1038/s41431-021-00892-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071842PMC
November 2021

A Patient with neonatal cholestasis.

J Mother Child 2021 Jul 16;24(4):31-33. Epub 2021 Jul 16.

Center for Metabolic Diseases, University Hospital Leuven, Leuven, Belgium.

The patient, a boy born in 1991, showed pronounced polyostotic fibrous dysplasia due to McCune-Albright syndrome, as well as Gilbert syndrome and Charcot-Marie-Tooth neuropathy caused by a mutation. In addition, the patient, his sister, mother and maternal grandfather had intermittently increased plasma arginine and lysine levels, most probably due to heterozygosity for a novel pathogenic variant.
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http://dx.doi.org/10.34763/jmotherandchild.20202404.d-20-00012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8330358PMC
July 2021

Impaired instrumental learning in Spred1 mice, a model for a rare RASopathy.

Genes Brain Behav 2021 06 4;20(5):e12727. Epub 2021 Mar 4.

Department of Human Genetics, KU Leuven, Leuven, Belgium.

RASopathies are neuro-cardio-facio-cutaneous disorders stemming from mutations in genes regulating the RAS-MAPK pathway. Legius syndrome is a rare RASopathy disorder caused by mutations in the SPRED1 gene. SPRED1 protein negatively regulates activation of Ras by inhibiting RAS/RAF and by its interaction with neurofibromin, a Ras GTPase-activating protein (RAS-GAP). Cognitive impairments have been reported in Legius syndrome as well as in other RASopathy disorders. Modelling these cognitive deficits in a Spred1 mouse model for Legius syndrome has demonstrated spatial learning and memory deficits, but other cognitive domains remained unexplored. Here, we attempted to utilize a cognitive touchscreen battery to investigate if Spred1 mice exhibit deficits in other cognitive domains. We show that Spred1 mice had heterogeneous performance in instrumental operant learning, with a large subgroup (n = 9/20) failing to reach the standard criterion on touchscreen operant pretraining, precluding further cognitive testing. To examine whether targeting the RAS-MAPK signalling pathway could rescue these cognitive impairments, Spred1 mice were acutely treated with the clinically relevant mitogen-activated protein kinase (MEK) inhibitor PD325901. However, MEK inhibition did not improve their instrumental learning. We conclude that Spred1 mice can model severe cognitive impairments that cannot be reversed in adulthood.
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http://dx.doi.org/10.1111/gbb.12727DOI Listing
June 2021

Comprehensive targeted next-generation sequencing approach in the molecular diagnosis of gastrointestinal stromal tumor.

Genes Chromosomes Cancer 2021 04 14;60(4):239-249. Epub 2020 Dec 14.

Department for Human Genetics, University Hospitals Leuven, KU Leuven, Leuven, Belgium.

Mutational analysis guides therapeutic decision making in patients with advanced-stage gastrointestinal stromal tumors (GISTs). We evaluated three targeted next-generation sequencing (NGS) assays, consecutively used over 4 years in our laboratory for mutational analysis of 162 primary GISTs: Agilent GIST MASTR, Illumina TruSight 26 and an in-house developed 96 gene panels. In addition, we investigated the feasibility of a more comprehensive approach by adding targeted RNA sequencing (Archer FusionPlex, 11 genes) in an attempt to reduce the number of Wild Type GISTs. We found KIT or PDGFRA mutations in 149 out of 162 GISTs (92.0%). Challenging KIT exon 11 alterations were initially missed by different assays in seven GISTs and typically represented deletions at the KIT intron 10-exon 11 boundary or large insertions/deletions (>24 base pairs). Comprehensive analysis led to the additional identification of driver alterations in 8/162 GISTs (4.9%): apart from BRAF and SDHA mutations (one case each), we found five GISTs harboring somatic neurofibromatosis type 1 (NF1) alterations (3.1%) and one case with an in-frame TRIM4-BRAF fusion not reported in GIST before. Eventually, no driver alteration was found in two out of 162 GISTs (1.2%) and three samples (1.9%) failed analysis. Our study shows that a comprehensive targeted NGS approach is feasible for routine mutational analysis of GIST, thereby substantially reducing the number of Wild Type GISTs, and highlights the need to optimize assays for challenging KIT exon 11 alterations.
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http://dx.doi.org/10.1002/gcc.22923DOI Listing
April 2021

Autism in neurofibromatosis type 1: misuse of covariance to dismiss autistic trait burden.

Dev Med Child Neurol 2021 02 20;63(2):233-234. Epub 2020 Aug 20.

Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA.

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http://dx.doi.org/10.1111/dmcn.14653DOI Listing
February 2021

Keratinocytic epidermal nevi associated with localized fibro-osseous lesions without hypophosphatemia.

Pediatr Dermatol 2020 Sep 14;37(5):890-895. Epub 2020 Jul 14.

Department of Dermatology, University Hospitals of Leuven, Leuven, Belgium.

Keratinocytic epidermal nevi (KEN) are characterized clinically by permanent hyperkeratosis in the distribution of Blaschko's lines and histologically by hyperplasia of epidermal keratinocytes. KEN with underlying RAS mutations have been associated with hypophosphatemic rickets and dysplastic bone lesions described as congenital cutaneous skeletal hypophosphatemia syndrome. Here, we describe two patients with keratinocytic epidermal nevi, in one associated with a papular nevus spilus, who presented with distinct localized congenital fibro-osseous lesions in the lower leg, diagnosed on both radiology and histology as osteofibrous dysplasia, in the absence of hypophosphatemia or rickets, or significantly raised FGF23 levels but with distinct mosaic HRAS mutations. This expands the spectrum of cutaneous/skeletal mosaic RASopathies and alerts clinicians to the importance of evaluating for bony disease even in the absence of bone profile abnormalities.
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http://dx.doi.org/10.1111/pde.14254DOI Listing
September 2020

Cancer Surveillance in Healthy Carriers of Germline Pathogenic Variants in : A Review of Secondary Prevention Guidelines.

J Oncol 2020 20;2020:9873954. Epub 2020 Jun 20.

Department of General Medical Oncology, University Hospitals Leuven, Leuven, Belgium.

Germline pathogenic alterations in the breast cancer susceptibility genes 1 () and 2 () are the most prevalent causes of hereditary breast and ovarian cancer. The increasing trend in proportion of cancer patients undergoing genetic testing, followed by predictive testing in families of new index patients, results in a significant increase of healthy germline 2 mutation carriers who are at increased risk for breast, ovarian, and other -related cancers. This review aims to give an overview of available screening guidelines for female and male carriers of pathogenic or likely pathogenic germline variants per cancer type, incorporating malignancies that are more or less recently well correlated with . We selected guidelines from national/international organizations and/or professional associations that were published or updated between January 1, 2015, and February 1, 2020. In total, 12 guidelines were included. This review reveals several significant discordances between the different guidelines. Optimal surveillance strategies depend on accurate age-specific cancer risk estimates, which are not reliably available for all -related cancers. Up-to-date national or international consensus guidelines are of utmost importance to harmonize counseling and proposed surveillance strategies for carriers.
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http://dx.doi.org/10.1155/2020/9873954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322604PMC
June 2020

Genetic basis of neurofibromatosis type 1 and related conditions, including mosaicism.

Childs Nerv Syst 2020 10 29;36(10):2285-2295. Epub 2020 Jun 29.

Department of Human Genetics, University of Leuven, Herestraat 49, 3000, Leuven, Belgium.

Introduction: Neurofibromatosis type 1 (NF1) is a frequent autosomal dominant disorder characterised by café-au-lait maculae (CALM), skinfold freckling, iris Lisch nodules and benign peripheral nerve sheath tumours (neurofibromas).

Mechanism: The NF1 gene is a tumour suppressor gene and NF1 individuals have an increased risk for a long list of tumours, all resulting from a second hit in the normal copy of the NF1 gene. Remarkably, some non-tumour phenotypes such as CALM and pseudarthrosis are also caused by a "second hit". Germline mutations inactivating the NF1 gene show a large variability in genetic mechanisms ranging from single-nucleotide substitutions and somatic mosaicism to large deletions affecting neighbouring genes. Molecular confirmation of the clinical diagnosis is becoming increasingly more important to differentiate NF1 from other syndromes such as Legius syndrome, to investigate genotype-phenotype correlations relevant in 10% of cases and to detect somatic mosaicism.

Surveillance And Therapy: Some degree of learning difficulties, attention deficit and social problems are observed in most children and affect quality of life. There is a large individual variability in complications and the evolution of the disease is difficult to predict. Specialised outpatient clinics for children have been widely established and are important for surveillance and guidance. Regular surveillance is also important for adolescents and adults because many tumour complications can be detected by whole-body MRI and treated even before symptoms develop and irreversible damage occurs. Recent data on nodular plexiform neurofibromas with continued growth in adolescents and young adults show that many of these tumours are premalignant lesions called atypical neurofibromatous neoplasm of uncertain biological potential (ANNUBP). Specific surveillance and timely local resection of these benign peripheral nerve sheath tumours might be important to prevent malignant degeneration. In the last years, targeted therapy with MEK inhibitors has shown promise to treat unresectable and symptomatic plexiform neurofibromas. Many more challenges remain to find the best way to monitor children and adults for potential complications and to find a satisfying cure for many complications in this disorder.
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http://dx.doi.org/10.1007/s00381-020-04771-8DOI Listing
October 2020

Legius Syndrome and its Relationship with Neurofibromatosis Type 1.

Acta Derm Venereol 2020 Mar;100(7):adv00093

Neurofibromatosis type 1 (NF1) is the most common disorder characterized by multiple café-au-lait macules. Most individuals with this autosomal dominant disorder also have other features, such as skinfold freckling, iris Lisch nodules and benign or malignant peripheral nerve sheath tumours. Legius syndrome is a less frequent autosomal dominant disorder with similar multiple café-au-lait macules and skinfold freckling. Legius syndrome is not characterized by an increased risk of tumours, and a correct diagnosis is important. In young children with a sporadic form of multiple café-au-lait macules with or without freckling and no other manifestations of NF1 these 2 conditions cannot be differentiated based on clinical examination. Molecular analysis of the NF1 and SPRED1 genes is usually needed to differentiate the 2 conditions. Other less frequent conditions with café-au-lait macules are Noonan syndrome with multiple lentigines, constitutional mismatch repair deficiency and McCune-Albright syndrome.
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http://dx.doi.org/10.2340/00015555-3429DOI Listing
March 2020

Next-generation sequencing in prenatal setting: Some examples of unexpected variant association.

Eur J Med Genet 2020 May 10;63(5):103875. Epub 2020 Feb 10.

Center for Human Genetics, University Hospitals Leuven, Catholic University Leuven, Leuven, Belgium. Electronic address:

The application of next-generation sequencing to fetal pathology has proved to increase the diagnostic yield in fetuses with abnormal ultrasounds. We retrospectively reviewed genetic data of 30 selected cases studied through targeted resequencing of OMIM genes. In our experience, clinical data proved to be essential to support diagnostic reasoning and enhance variants' assessment. The molecular diagnosis was reached in 19/30 (63%) cases. Only in 7/19 cases the molecular diagnosis confirmed the initial diagnostic hypothesis, showing the relevance of the genotype-first approach. According to the genotype-phenotype correlation, we were able to divide the solved cases into three groups: i) the correlation is well established but it was missed due to lack of specificity, unusual presentation or recent description; ii) the clinical presentation is much more severe than currently known for the underlying condition; iii) the correlation does not recapitulate the entire phenotype, possibly due to the fetal presentation or multiple coexisting conditions. Moreover, we found a higher proportion of recessive diagnosis in abnormal fetuses compared to cohorts of individuals with developmental delay. Our findings suggest that fetal pathology may be enriched in rare alleles and/or in unusual combinations, counter-selected in postnatal genomes and thus contributing to both phenotypic extremeness and atypical presentation.
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http://dx.doi.org/10.1016/j.ejmg.2020.103875DOI Listing
May 2020

Noninvasive prenatal diagnosis by genome-wide haplotyping of cell-free plasma DNA.

Genet Med 2020 05 6;22(5):962-973. Epub 2020 Feb 6.

Center for Human Genetics, KU Leuven, Leuven, Belgium.

Purpose: Whereas noninvasive prenatal screening for aneuploidies is widely implemented, there is an increasing need for universal approaches for noninvasive prenatal screening for monogenic diseases. Here, we present a cost-effective, generic cell-free fetal DNA (cffDNA) haplotyping approach to scan the fetal genome for the presence of inherited monogenic diseases.

Methods: Families participating in the preimplantation genetic testing for monogenic disorders (PGT-M) program were recruited for this study. Two hundred fifty thousand single-nucleotide polymorphisms (SNPs) captured from maternal plasma DNA along with genomic DNA from family members were massively parallel sequenced. Parental genotypes were phased via an available genotype from a close relative, and the fetal genome-wide haplotype and copy number were determined using cffDNA haplotyping analysis based on estimation and segmentation of fetal allele presence in the maternal plasma.

Results: In all families tested, mutational profiles from cffDNA haplotyping are consistent with embryo biopsy profiles. Genome-wide fetal haplotypes are on average 97% concordant with the newborn haplotypes and embryo haplotypes.

Conclusion: We demonstrate that genome-wide targeted capture and sequencing of polymorphic SNPs from maternal plasma cell-free DNA (cfDNA) allows haplotyping and copy-number profiling of the fetal genome during pregnancy. The method enables the accurate reconstruction of the fetal haplotypes and can be easily implemented in clinical practice.
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http://dx.doi.org/10.1038/s41436-019-0748-yDOI Listing
May 2020

Examination of the genetic factors underlying the cognitive variability associated with neurofibromatosis type 1.

Genet Med 2020 05 4;22(5):889-897. Epub 2020 Feb 4.

Department of Neuroscience, Erasmus Medical Center, Rotterdam, The Netherlands.

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder associated with cognitive deficits. The NF1 cognitive phenotype is generally considered to be highly variable, possibly due to the observed T2-weighted hyperintensities, loss of heterozygosity, NF1-specific genetic modifiers, or allelic imbalance.

Methods: We investigated cognitive variability and assessed the contribution of genetic factors by performing a retrospective cohort study and a monozygotic twin case series. We included data of 497 children with genetically confirmed NF1 and an IQ assessment, including 12 monozygotic twin and 17 sibling sets.

Results: Individuals carrying an NF1 chromosomal microdeletion showed significant lower full-scale IQ (FSIQ) scores than individuals carrying intragenic pathogenic NF1 variants. For the intragenic subgroup, the variability in cognitive ability and the correlation of IQ between monozygotic NF1 twin pairs or between NF1 siblings is similar to the general population.

Conclusions: The variance and heritability of IQ in individuals with NF1 are similar to that of the general population, and hence mostly driven by genetic background differences. The only factor that significantly attenuates IQ in NF1 individuals is the NF1 chromosomal microdeletion genotype. Implications for clinical management are that individuals with intragenic NF1 variants that score <1.5-2 SD below the mean of the NF1 population should be screened for additional causes of cognitive disability.
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http://dx.doi.org/10.1038/s41436-020-0752-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200599PMC
May 2020

The clinical relevance of intragenic deletions.

J Med Genet 2020 05 13;57(5):347-355. Epub 2020 Jan 13.

Department of Human Genetics, KU Leuven, Leuven, Belgium

Background: Intragenic deletions are susceptibility variants for neurodevelopmental disorders; however, their clinical interpretation is often unclear. Therefore, a literature study and an analysis of 43 previously unpublished deletions are provided.

Methods: The literature cohort covered 629 heterozygous deletions: 148 in controls, 341 in probands and 140 in carrier relatives, and was used for clinical hypothesis testing. Exact breakpoint determination was performed for 43 in-house deletions.

Results: The prevalence of exonic deletions in controls was ~1/3000 as compared with ~1/800 in patients with neurodevelopmental/neuropsychiatric disorders. The differential distribution of deletions across the gene between controls and probands allowed to distinguish distinct areas within the gene. Exon 6-24 deletions appeared only twice in over 100000 control individuals, had an estimated penetrance for neurodevelopmental disorders of 32.43%, a rate of 50% and segregated mainly with intellectual disability (ID) and schizophrenia. In contrast, exon 1-5 deletions appeared in 20 control individuals, had an estimated penetrance of 12.59%, a rate of 32.5% and were reported with a broad range of neurodevelopmental phenotypes. Exact breakpoint determination revealed six recurrent intron 5 deletions.

Conclusion: Exon 6-24 deletions have a high penetrance and are mainly associated with ID and schizophrenia. In contrast, the actual contribution of exon 1-5 deletions to a neurodevelopmental/neuropsychiatric disorder in an individual patient and family remains very difficult to assess. To enhance the clinical interpretation, this study provides practical considerations for counselling and an interactive table for comparing a deletion of interest with the available literature data.
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http://dx.doi.org/10.1136/jmedgenet-2019-106448DOI Listing
May 2020

Advancing RAS/RASopathy therapies: An NCI-sponsored intramural and extramural collaboration for the study of RASopathies.

Am J Med Genet A 2020 04 8;182(4):866-876. Epub 2020 Jan 8.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.
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http://dx.doi.org/10.1002/ajmg.a.61485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456498PMC
April 2020

NF1 patient missense variants predict a role for ATM in modifying neurofibroma initiation.

Acta Neuropathol 2020 01 29;139(1):157-174. Epub 2019 Oct 29.

Department of Experimental Hematology and Cancer Biology, Cincinati Children's Hospital Medical Center and Department of Pediatrics, University of Cincinanti School of Medicine, Cincinnati, OH, USA.

In Neurofibromatosis type 1, NF1 gene mutations in Schwann cells (SC) drive benign plexiform neurofibroma (PNF), and no additional SC changes explain patient-to-patient variability in tumor number. Evidence from twin studies suggests that variable expressivity might be caused by unidentified modifier genes. Whole exome sequencing of SC and fibroblast DNA from the same resected PNFs confirmed biallelic SC NF1 mutations; non-NF1 somatic SC variants were variable and present at low read number. We identified frequent germline variants as possible neurofibroma modifier genes. Genes harboring variants were validated in two additional cohorts of NF1 patients and by variant burden test. Genes including CUBN, CELSR2, COL14A1, ATR and ATM also showed decreased gene expression in some neurofibromas. ATM-relevant DNA repair defects were also present in a subset of neurofibromas with ATM variants, and in some neurofibroma SC. Heterozygous ATM G2023R or homozygous S707P variants reduced ATM protein expression in heterologous cells. In mice, genetic Atm heterozygosity promoted Schwann cell precursor self-renewal and increased tumor formation in vivo, suggesting that ATM variants contribute to neurofibroma initiation. We identify germline variants, rare in the general population, overrepresented in NF1 patients with neurofibromas. ATM and other identified genes are candidate modifiers of PNF pathogenesis.
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http://dx.doi.org/10.1007/s00401-019-02086-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243727PMC
January 2020

Clinical spectrum of individuals with pathogenic NF1 missense variants affecting p.Met1149, p.Arg1276, and p.Lys1423: genotype-phenotype study in neurofibromatosis type 1.

Hum Mutat 2020 01 26;41(1):299-315. Epub 2019 Oct 26.

Department of Dermatology and Venereology, Policlinico Umberto I, Sapienza University of Rome, Rome, Italy.

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population.
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http://dx.doi.org/10.1002/humu.23929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973139PMC
January 2020

Mutational spectrum by phenotype: panel-based NGS testing of patients with clinical suspicion of RASopathy and children with multiple café-au-lait macules.

Clin Genet 2020 02 12;97(2):264-275. Epub 2019 Dec 12.

Hereditary Cancer Group, Program of Predictive and Personalized Medicine of Cancer (PMPPC), Germans Trias & Pujol Research Institute (IGTP), Barcelona, Spain.

Children with neurofibromatosis type 1 (NF1) may exhibit an incomplete clinical presentation, making difficult to reach a clinical diagnosis. A phenotypic overlap may exist in children with other RASopathies or with other genetic conditions if only multiple café-au-lait macules (CALMs) are present. The syndromes that can converge in these inconclusive phenotypes have different clinical courses. In this context, an early genetic testing has been proposed to be clinically useful to manage these patients. We present the validation and implementation into diagnostics of a custom NGS panel (I2HCP, ICO-IMPPC Hereditary Cancer Panel) for testing patients with a clinical suspicion of a RASopathy (n = 48) and children presenting multiple CALMs (n = 102). We describe the mutational spectrum and the detection rates identified in these two groups of individuals. We identified pathogenic variants in 21 out of 48 patients with clinical suspicion of RASopathy, with mutations in NF1 accounting for 10% of cases. Furthermore, we identified pathogenic mutations mainly in the NF1 gene, but also in SPRED1, in more than 50% of children with multiple CALMs, exhibiting an NF1 mutational spectrum different from a group of clinically diagnosed NF1 patients (n = 80). An NGS panel strategy for the genetic testing of these two phenotype-defined groups outperforms previous strategies.
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http://dx.doi.org/10.1111/cge.13649DOI Listing
February 2020

CTCF variants in 39 individuals with a variable neurodevelopmental disorder broaden the mutational and clinical spectrum.

Genet Med 2019 12 26;21(12):2723-2733. Epub 2019 Jun 26.

Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA.

Purpose: Pathogenic variants in the chromatin organizer CTCF were previously reported in seven individuals with a neurodevelopmental disorder (NDD).

Methods: Through international collaboration we collected data from 39 subjects with variants in CTCF. We performed transcriptome analysis on RNA from blood samples and utilized Drosophila melanogaster to investigate the impact of Ctcf dosage alteration on nervous system development and function.

Results: The individuals in our cohort carried 2 deletions, 8 likely gene-disruptive, 2 splice-site, and 20 different missense variants, most of them de novo. Two cases were familial. The associated phenotype was of variable severity extending from mild developmental delay or normal IQ to severe intellectual disability. Feeding difficulties and behavioral abnormalities were common, and variable other findings including growth restriction and cardiac defects were observed. RNA-sequencing in five individuals identified 3828 deregulated genes enriched for known NDD genes and biological processes such as transcriptional regulation. Ctcf dosage alteration in Drosophila resulted in impaired gross neurological functioning and learning and memory deficits.

Conclusion: We significantly broaden the mutational and clinical spectrum ofCTCF-associated NDDs. Our data shed light onto the functional role of CTCF by identifying deregulated genes and show that Ctcf alterations result in nervous system defects in Drosophila.
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http://dx.doi.org/10.1038/s41436-019-0585-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892744PMC
December 2019

Maternal copy-number variations in the DMD gene as secondary findings in noninvasive prenatal screening.

Genet Med 2019 12 14;21(12):2774-2780. Epub 2019 Jun 14.

Center for Human Genetics, University Hospitals Leuven, Leuven, Belgium.

Purpose: Noninvasive prenatal screening (NIPS) using genome sequencing also reveals maternal copy-number variations (CNVs). Those CNVs can be clinically actionable or harmful to the fetus if inherited. CNVs in the DMD gene potentially causing dystrophinopathies are among the most commonly observed maternal CNVs. We present our experience with maternal DMD gene CNVs detected by NIPS.

Methods: We analyzed the data of maternal CNVs detected in the DMD gene revealed by NIPS.

Results: Of 26,123 NIPS analyses, 16 maternal CNVs in the DMD gene were detected (1/1632 pregnant women). Variant classification regarding pathogenicity and phenotypic severity was based on public databases, segregation analysis in the family, and prediction of the effect on the reading frame. Ten CNVs were classified as pathogenic, four as benign, and two remained unclassified.

Conclusion: NIPS leverages CNV screening in the general population of pregnant women. We implemented a strategy for the interpretation and the return of maternal CNVs in the DMD gene detected by NIPS.
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http://dx.doi.org/10.1038/s41436-019-0564-4DOI Listing
December 2019

The sudden death of the combined first trimester aneuploidy screening, a single centre experience in Belgium.

Clin Chem Lab Med 2019 Oct;57(11):e294-e297

Clinical Department of Laboratory Medicine, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium, Phone: 003216347015, Fax: 003216347931.

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http://dx.doi.org/10.1515/cclm-2019-0231DOI Listing
October 2019

Neurofibromatosis type 1-related pseudarthrosis: Beyond the pseudarthrosis site.

Hum Mutat 2019 10 18;40(10):1760-1767. Epub 2019 Jun 18.

Department of Human Genetics, KU Leuven-University of Leuven, Leuven, Belgium.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting approximately 1 in 2,000 newborns. Up to 5% of NF1 patients suffer from pseudarthrosis of a long bone (NF1-PA). Current treatments are often unsatisfactory, potentially leading to amputation. To gain more insight into the pathogenesis we cultured cells from PA tissue and normal-appearing periosteum of the affected bone for NF1 mutation analysis. PA cells were available from 13 individuals with NF1. Biallelic NF1 inactivation was identified in all investigated PA cells obtained during the first surgery. Three of five cases sampled during a later intervention showed biallelic NF1 inactivation. Also, in three individuals, we examined periosteum-derived cells from normal-appearing periosteum proximal and distal to the PA. We identified the same biallelic NF1 inactivation in the periosteal cells outside the PA region. These results indicate that NF1 inactivation is required but not sufficient for the development of NF1-PA. We observed that late-onset NF1-PA occurs and is not always preceded by congenital bowing. Furthermore, the failure to identify biallelic inactivation in two of five later interventions and one reintervention with a known somatic mutation indicates that NF1-PA can persist after the removal of most NF1 negative cells.
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http://dx.doi.org/10.1002/humu.23783DOI Listing
October 2019

First International Conference on RASopathies and Neurofibromatoses in Asia: Identification and advances of new therapeutics.

Am J Med Genet A 2019 06 25;179(6):1091-1097. Epub 2019 Mar 25.

MEDGENOME Labs, Bangalore, India.

The neurofibromatoses, which include neurofibromatosis type I (NF1), neurofibromatosis type II (NF2), and schwannomatosis, are a group of syndromes characterized by tumor growth in the nervous system. The RASopathies are a group of syndromes caused by germline mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. The RASopathies include NF1, Noonan syndrome, Noonan syndrome with multiple lentigines, Costello syndrome, cardio-facio-cutaneous syndrome, Legius syndrome, capillary malformation arterio-venous malformation syndrome, and SYNGAP1 autism. Due to their common underlying pathogenetic etiology, all these syndromes have significant phenotypic overlap of which one common feature include a predisposition to tumors, which may be benign or malignant. Together as a group, they represent one of the most common multiple congenital anomaly syndromes estimating to affect approximately one in 1000 individuals worldwide. The subcontinent of India represents one of the largest populations in the world, yet remains underserved from an aspect of clinical genetics services. In an effort to bridge this gap, the First International Conference on RASopathies and Neurofibromatoses in Asia: Identification and Advances of New Therapeutics was held in Kochi, Kerala, India. These proceedings chronicle this timely and topical international symposium directed at discussing the best practices and therapies for individuals with neurofibromatoses and RASopathies.
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http://dx.doi.org/10.1002/ajmg.a.61125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279388PMC
June 2019

From process to progress-2017 International Conference on Neurofibromatosis 1, Neurofibromatosis 2 and Schwannomatosis.

Am J Med Genet A 2019 06 25;179(6):1098-1106. Epub 2019 Mar 25.

Division of Neuroscience, Oregon National Primate Research Center, and Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, Oregon.

The neurofibromatoses are inherited, tumor suppressor disorders that are characterized by multiple, benign peripheral nerve sheath tumors and other nervous system tumors. Each disease is associated with a distinct genetic mutation and with a different pathogenesis and clinical course. Neurofibromatosis 1 (NF1) is common and epitomized by multiple neurofibromas with widespread complications. NF2 and schwannomatosis are rare diseases that are typified by multiple schwannomas that are particularly painful in people with schwannomatosis. Since 1985, the Children's Tumor Foundation (formerly the National Neurofibromatosis Foundation) has hosted an international Neurofibromatosis Conference, bringing together international participants who are focused on NF research and clinical care. The 2017 Conference, held in Washington, DC, was among the largest gatherings of NF researchers to date and included presentations from clinicians and basic scientists, highlighting new data regarding the molecular and cellular mechanisms underlying each of these diseases as well as results from clinical studies and clinical trials. This article summarizes the findings presented at the meeting and represents the current state-of-the art for NF research.
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http://dx.doi.org/10.1002/ajmg.a.61112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488427PMC
June 2019
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