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STUDY QUESTIONCan long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents?SUMMARY ANSWERLong-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs.WHAT IS KNOWN ALREADYUniversal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype.STUDY DESIGN, SIZE, DURATIONThis is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles.PARTICIPANTS/MATERIALS, SETTING, METHODSTo identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5–10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping.MAIN RESULTS AND THE ROLE OF CHANCEThe parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT.LIMITATIONS, REASONS FOR CAUTIONThe main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs.WIDER IMPLICATIONS OF THE FINDINGSTargeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 ‘Methods for haplotyping single-cells’ and ZL913096-PCT/EP2014/068315-WO/2015/028576 ‘Haplotyping and copy number typing using polymorphic variant allelic frequencies’ licensed to Agilent Technologies. All other authors have no conflict of interest to declare.TRIAL REGISTRATION NUMBERN/A.

Supplementary Figure 1 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Neurofibromatosis type 1 (NF1) is a common disorder that arises secondary to mutations in the tumor suppressor gene NF1. Glomus tumors are small, benign but painful tumors that originate from the glomus body, a thermoregulatory shunt concentrated in the fingers and toes. We report 11 individuals with NF1 who harbored 20 glomus tumors of the fingers and 1 in the toe; 5 individuals had multiple glomus tumors. We hypothesized that biallelic inactivation of NF1 underlies the pathogenesis of these tumors. In 12 NF1-associated glomus tumors, we used cell culture and laser capture microdissection to isolate DNA. We also analyzed two sporadic (not NF1-associated) glomus tumors. Genetic analysis showed germ line and somatic NF1 mutations in seven tumors. RAS mitogen-activated protein kinase hyperactivation was observed in cultured NF1−/− glomus cells, reflecting a lack of inhibition of the pathway by functional neurofibromin, the protein product of NF1. No abnormalities in NF1 or RAS mitogen-activated protein kinase activation were found in sporadic glomus tumors. By comparative genomic hybridization, we observed amplification of the 3′-end of CRTAC1 and a deletion of the 5′-end of WASF1 in two NF1-associated glomus tumors. For the first time, we show that loss of neurofibromin function is crucial in the pathogenesis of glomus tumors in NF1. Glomus tumors of the fingers or toes should be considered as part of the tumor spectrum of NF1. [Cancer Res 2009;69(18):7393–401]

Supplementary Figure Legend from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Supplementary Table 1 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Supplementary Table 2 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Genotype; Cancer phenotypes; Genetic tumour Genotip; Fenotips del càncer; Tumor genètic Genotipo; Fenotipos del cáncer; Tumor genético Background Truncating pathogenic or likely pathogenic variants of CDH1 cause hereditary diffuse gastric cancer (HDGC), a tumour risk syndrome that predisposes carrier individuals to diffuse gastric and lobular breast cancer. Rare CDH1 missense variants are often classified as variants of unknown significance. We conducted a genotype–phenotype analysis in families carrying rare CDH1 variants, comparing cancer spectrum in carriers of pathogenic or likely pathogenic variants (PV/LPV; analysed jointly) or missense variants of unknown significance, assessing the frequency of families with lobular breast cancer among PV/LPV carrier families, and testing the performance of lobular breast cancer-expanded criteria for CDH1 testing. Methods This genotype-first study used retrospective diagnostic and clinical data from 854 carriers of 398 rare CDH1 variants and 1021 relatives, irrespective of HDGC clinical criteria, from 29 institutions in ten member-countries of the European Reference Network on Tumour Risk Syndromes (ERN GENTURIS). Data were collected from Oct 1, 2018, to Sept 20, 2022. Variants were classified by molecular type and clinical actionability with the American College of Medical Genetics and Association for Molecular Pathology CDH1 guidelines (version 2). Families were categorised by whether they fulfilled the 2015 and 2020 HDGC clinical criteria. Genotype–phenotype associations were analysed by Student's t test, Kruskal-Wallis, χ2, and multivariable logistic regression models. Performance of HDGC clinical criteria sets were assessed with an equivalence test and Youden index, and the areas under the receiver operating characteristic curves were compared by Z test. Findings From 1971 phenotypes (contributed by 854 probands and 1021 relatives aged 1–93 years), 460 had gastric and breast cancer histology available. CDH1 truncating PV/LPVs occurred in 176 (21%) of 854 families and missense variants of unknown significance in 169 (20%) families. Multivariable logistic regression comparing phenotypes occurring in families carrying PV/LPVs or missense variants of unknown significance showed that lobular breast cancer had the greatest positive association with the presence of PV/LPVs (odds ratio 12·39 [95% CI 2·66–57·74], p=0·0014), followed by diffuse gastric cancer (8·00 [2·18–29·39], p=0·0017) and gastric cancer (7·81 [2·03–29·96], p=0·0027). 136 (77%) of 176 families carrying PV/LPVs fulfilled the 2015 HDGC criteria. Of the remaining 40 (23%) families, who did not fulfil the 2015 criteria, 11 fulfilled the 2020 HDGC criteria, and 18 had lobular breast cancer only or lobular breast cancer and gastric cancer, but did not meet the 2020 criteria. No specific CDH1 variant was found to predispose individuals specifically to lobular breast cancer, although 12 (7%) of 176 PV/LPV carrier families had lobular breast cancer only. Addition of three new lobular breast cancer-centred criteria improved testing sensitivity while retaining high specificity. The probability of finding CDH1 PV/LPVs in patients fulfilling the lobular breast cancer-expanded criteria, compared with the 2020 criteria, increased significantly (AUC 0·92 vs 0·88; Z score 3·54; p=0·0004). Interpretation CDH1 PV/LPVs were positively associated with HDGC-related phenotypes (lobular breast cancer, diffuse gastric cancer, and gastric cancer), and no evidence for a positive association with these phenotypes was found for CDH1 missense variants of unknown significance. CDH1 PV/LPVs occurred often in families with lobular breast cancer who did not fulfil the 2020 HDGC criteria, supporting the expansion of lobular breast cancer-centred criteria. Funding European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme. European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme.

Objective Combined immunohistochemical and molecular classification using the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) independently predicts prognosis in endometrial carcinoma (EC). As next-generation sequencing (NGS) is entering clinical practice, we evaluated whether more comprehensive immunomolecular profiling (CIMP), including NGS and extended immunohistochemical analysis, could further refine the current ProMisE classification. Methods A series of 120 consecutive ECs, classified according to ProMisE, was stained immunohistochemically for CD3, CD8, PD-L1, beta-catenin and L1CAM. An in-house 96 gene NGS panel was performed on a subset of 44 ECs, representing the 4 ProMisE subgroups (DNA polymerase epsilon catalytic subunit exonuclease domain mutated (POLEmut), mismatch repair deficient (MMRd), p53 abnormal (p53 abn) and no specific molecular profile (NSMP) ECs). Cases harboring non-hotspot POLE variants were analyzed with Illumina TruSight Oncology 500 NGS panel (TSO500) as a surrogate for whole-exome sequencing. Results Eight cases harbored POLE variants, half of which were hotspots. Using TSO500, non-hotspot POLE variants were classified as pathogenic (3) or variant of unknown significance (1). POLEmut and MMRd ECs typically showed higher numbers of CD3+/CD8+ tumor-infiltrating lymphocytes and higher PD-L1 expression in tumor-infiltrating immune cells. p53 abn ECs showed significantly higher L1CAM immunoreactivity and frequently harbored gene amplifications including HER2 (25%), but typically lacked ARID1A or PTEN variants. Beta-catenin-positivity and FGFR2 variants were predominantly found in NSMP ECs. Conclusions Our data show that CIMP adds significant value to EC characterization and may help to determine pathogenicity of non-hotspot POLE variants, encountered more frequently than expected in our series. In addition, CIMP may reveal ECs benefitting from immune checkpoint inhibition and allows upfront identification of targetable alterations, such as HER2 amplification in p53 abn ECs.

In recent years, immune checkpoint inhibitors (ICPI) have become widely used for multiple solid malignancies. Reliable predictive biomarkers for selection of patients who would benefit most are lacking. Several tumor types with somatic or germline alterations in genes involved in the DNA damage response (DDR) pathway harbor a higher tumor mutational burden, possibly associated with an increased tumoral neoantigen load. These neoantigens are thought to lead to stronger immune activation and enhanced response to ICPIs. We present a series of seven patients with different malignancies with germline disease-associated variants in DDR genes (BRCA1, BRCA2, CHEK2) responding favorably to ICPIs. ispartof: Curr Oncol vol:28 issue:5 pages:3227-3239 ispartof: location:Switzerland status: published

BackgroundMalignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise from the peripheral nervous system. Half of the tumors develop in the context of the genetic disease Neurofibromatosis type 1 (NF1) and the rest are sporadic sarcomas. MPNSTs have a dismal prognosis due to their aggressiveness and tendency to metastasize, and new treatment options are needed. The diagnosis of MPNSTs can be challenging, especially outside of the NF1 context since specific histological criteria have not been completely established. Genomic analysis may both facilitate differential diagnoses and suggest precision medicine strategies.MethodsWe generated a complete genomic resource of a set of widely used human NF1-related and sporadic MPNST cell lines by applying ploidy analysis, whole genome and whole exome sequencing and SNP-array analysis, complemented by methylome-based classification and immunofluorescence of cell identity markers (SOX9, SOX10, S100B).ResultsNF1 MPNST cell lines faithfully recapitulated the genomic copy number profile of primary MPNSTs. Structural variants were key players in the complete inactivation of most recurrently altered tumor suppressor genes (TSGs) (NF1, CDKN2A, SUZ12/EED), while small variants played a minor role in the NF1 context, both concerning TSG inactivation and due to the absence of gain-of-function mutations. In clear contrast, the sporadic cell lines (STS-26T, HS-Sch-2, HS-PSS) did not recapitulate the copy number profile of primary MPNSTs. They carried different TSG inactivation and exhibited gain-of-function mutations by predicted kinase activation or generation of fusion genes. Mutational frequencies and signatures emerged as promising informative tools for aiding in MPNST differential diagnosis. Due to the multiple genomic differences exhibited, we complemented their characterization using a methylome-based classifier. All NF1-related cell lines were assigned within the MPNST group, while sporadic cell lines clustered either with melanomas or with an uncertain MPNST-like sarcoma group. The staining of cell identity markers reinforced the idea of a potential misdiagnose of the MPNSTs used to derive the sporadic cell lines analyzed.ConclusionsDeep genomic analysis, together with methylome-based sarcoma classification and cell identity marker analysis, challenged the MPNST identity of sporadic cell lines. Results presented here open an opportunity to revise MPNST differential diagnosis and classification.

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. ispartof: PEDIATRIC DERMATOLOGY vol:37 issue:5 pages:890-895 ispartof: location:United States status: published

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

BACKGROUND: Pathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study. METHODS: Information was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age. RESULTS: At time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5-3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1-2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1-3.5), with trends up to 2-4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3-0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4-0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2. CONCLUSION: The PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants. ispartof: EUROPEAN JOURNAL OF MEDICAL GENETICS vol:65 issue:12 ispartof: location:Netherlands status: published

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.

Congenital melanocytic nevus syndrome (CMNS) is a rare condition characterized by pigmented skin lesions that is usually present at birth and is associated with an increased risk of neurological abnormalities and malignant melanoma. It mostly results from a post-zygotic NRAS mutation of neural-derived crest cells, leading to an uncontrolled cell growth. Due to the increased knowledge of the genetics underlying CMNS, targeted therapy becomes a promising treatment option. We present a case of CMNS in a newborn. Physical examination at birth showed a giant congenital melanocytic nevus, extending from the occipital to the lower lumbar region. An MRI-scan revealed multiple cerebral and cerebellar parenchymal lesions. Genetic analysis of the cutaneous lesions showed the presence of a NRAS Q61R mutation. The patient was treated with dermabrasion to reduce the color intensity of the nevus. However, this was complicated with recurrent wound infections and laborious wound healing. At the age of 1 year, the patient had an age-appropriate psychomotor development, without neurological deficits. This article is protected by copyright. All rights reserved. ispartof: Journal Of Cutaneous Pathology vol:48 issue:12 pages:1497-1503 ispartof: location:United States status: Published online

Background: The introduction of multi-gene panel testing and improved awareness under patients and physicians has led to an increase of individuals with known germline pathogenic variants in hereditary breast and ovarian cancer (HBOC) genes. Significant regional differences exist in germline mutational landscape. We aimed to report the findings from multi-gene panel testing in a large Belgian cohort of individuals at risk for HBOC. Methods: All individuals who underwent multi-gene panel testing for HBOC at the Center for Human Genetics of the University Hospitals Leuven since the introduction of the panel were included (March 2016-April 2019). All included individuals were considered candidates for HBOC-panel testing by the requesting physician based on a personal or familial history of breast and/or ovarian cancer. Testing criteria from the Belgian Society of Human Genetics (www.beshg.be/download/guidelines/Guidelines_HBOC_2018.pdf) were met in the vast majority. The panel used was the BRCA Hereditary Cancer MASTR Plus® (Agilent, Belgium), with sequencing of BARD1, BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, TP53, MRE11A, RAD50, NBN, FAM175A, ATM, PALB2, STK11, MEN1, PTEN, CDH1, MUTYH, CHEK2, BLM, XRCC2, EPCAM, MLH1, MSH6, PMS2 and MSH2. Sequencing was performed by NGS on a Miseq platform (Illumina). Genomic deletions and duplications in BRCA1 and BRCA2 were investigated with multiplex ligation-dependent probe amplification. We hereby report on the frequency of pathogenic and likely pathogenic germline variants in this population. Results: In 5422 individuals who underwent multi-gene panel testing, we detected 665 pathogenic or likely pathogenic variants in 639 patients (11,7%). In 25 patients (0.46%), more than one relevant alteration was detected with double heterozygosity in 24 individuals and triple heterozygosity in one. Germline variants in BRCA1 and BRCA2 were detected in 178 (3.3%) and 144 (2.7%) patients, resulting in a fraction of 26,4% and 21,4% of detected variants respectively. Relevant alterations in CHEK2, ATM, PALB2 and TP53 were observed in 135 (2.5%), 93 (1.7%), 26 (0.5%) and 11 (0.2%) patients respectively, accounting together for 39.3% of detected variants. Alterations in BRIP1/RAD51C/RAD51D were retrieved in 64 patients (1.2%) and alterations in mismatch-repair genes MSH6/MLH1/PMS2/MSH2 were detected in 0.3% of patients. These patients where dominantly referred for familial history of ovarian cancer. Furthermore, germline alterations in PTEN, CDH1 and BLM were observed in 3, 2 and 1 cases respectively. Double heterozygosity for ATM+CHEK2 and for ATM+BRCA2 were both observed in 3 cases. In the patient with triple heterozygosity, co-occurrence of pathogenic variants in BRCA2, ATM and CHEK2 was detected. Conclusions: In a large Belgian cohort of 5422 individuals at risk for HBOC who underwent multi-gene panel testing, a pathogenic or likely pathogenic germline variant was detected in 11,7% of patients, and in 0,46% of patients double or triple heterozygosity for HBOC-variants was observed. Almost 40% of detected variants were alterations beyond BRCA correlated with hereditary breast cancer (CHEK2, ATM, PALB2 and TP53). Given the time-lag to predictive testing in families, a significant rise in healthy carriers with these non-BRCA alterations is expected in the upcoming years. Citation Format: Kevin Punie, Griet Hoste, Griet Van Buggenhout, Ellen Denayer, Hilde Brems, Hilde Peeters, Ann Smeets, Ines Nevelsteen, Patrick Neven, Jan Ardui, Renate Prevos, Machteld Keupers, Chantal Van Ongeval, Giuseppe Floris, Christine Desmedt, Hans Wildiers, Geneviève Michils, Hilde Van Esch, Eric Legius. Germline mutational landscape in 5422 individuals at risk for hereditary breast and ovarian cancer who underwent multi-gene panel testing [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-03.

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.

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 DNM2 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 SLC7A2 variant.

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.

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.

OBJECTIVES: To apply the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) to a consecutive series of endometrial cancer (EC) patients diagnosed at a tertiary referral center and assign EC specimens to one of four molecular subgroups using immunohistochemistry (IHC) for p53/mismatch repair protein expression and sequencing for Polymerase Epsilon Exonuclease Domain Mutations (POLE-EDM). Mismatch Repair Deficient (MMR-D) cases were more thoroughly investigated to identify underlying somatic or germline genetic defects. METHODS: Hundred-and eight consecutive endometrial cancer patients, diagnosed between March 2017 and April 2019, were subjected to immunohistochemical and molecular analysis, according to ProMisE. IHC for p53 and the mismatch repair proteins (MLH1, PMS2, MSH6 and PMS2) was performed. All patients were also tested for POLE-EDM by Sanger sequencing. In addition, tumor and corresponding normal tissue of cases with abnormal MMR IHC were tested by PCR for microsatellite instability (MSI) (MSI analysis system, Promega). Hypermethylation of MLH1 promotor was tested with (methylation specific) multiplex ligation dependent probe amplification. MMR-D cases were subjected to germline mutation analysis of the mismatch repair genes, using next generation sequencing on MiSeq (Illumina) with the BRCA Hereditary Cancer MASTR Plus, (Multiplicom/Agilent), RNA mutation analysis and MLPA. RESULTS: FIGO classification was stage IA (n = 54), IB (n = 22) II(n = 8), III(n = 18) and IV(n = 6). Of the 33 patients with MMR-D on IHC (31%), 26 showed MLH1 promotor hypermethylation as the probable cause of MMR-D. The remaining 7 patients without MLH1 promotor hypermethylation were referred for germline analysis of Lynch syndrome. Six patients carried a pathogenic germline mutation in one of the mismatch repair genes: MSH6(n = 3), PMS2(n = 1), MLH1(n = 1) and MSH2 (n = 1). Pathogenic POLE-EDM were identified in 7 (6%) patients. Multiple molecular features (POLE-EDM + MMR-D or POLE-EDM + p53 abnormal) were observed in 4 patients (4%). A high concordance between MMR-D and microsatellite instability was observed in our cohort. In cases of a genetic defect in the MMR genes, we do note a large proportion of cases exhibiting microsatellite instability. On the contrary a hypermutation state, as seen in POLE EDM, does not result in accompanied phenotypic changes in MSI status. CONCLUSION: The ProMisE classification proved to be an efficient and easily implementable system. Future research should elucidate the precise biological and prognostic meaning of the cases with multiple molecular markers. ispartof: GYNECOLOGIC ONCOLOGY vol:157 issue:1 pages:245-251 ispartof: location:United States status: published

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

As a multisystem developmental disorder, neurofibromatosis type I can affect many body systems. The overall rate of congenital anomalies identified in NF1 appears to be increased, with a best estimate of a three-fold increase compared to the general population (Leppavirta et al., Orphanet J Rare Dis. 13:5, 2018). Aside from bony dysplasia or plexiform neurofibromas apparent from birth, much of this increase is attributable to congenital heart disease. This chapter focusses on manifestations of NF1 in organ systems other than the central and peripheral nervous systems and those described in Chaps. 5– 7. Cardiovascular problems are common, and an important cause of morbidity and mortality in individuals with NF1. Hypertension is increased in prevalence, with renal artery stenosis (with or without other vascular predisposing factors) and phaeochromocytoma as important NF1-associated causes. Interstitial pulmonary disease is much less common than cardiac disease, but can be associated with a poor prognosis. Gastrointestinal (GI) manifestations of NF1 are common, and troublesome for many patients, including abnormalities of the enteric nervous system causing motility problems, and epithelial or mesenchymal tumours of the GI tract. Haematological manifestations of NF1 are also recognised; in keeping with overall tumour predisposition (Chap. 17), leukaemia, particularly myelomonocytic leukaemia, is a significant cause of very early mortality in children with NF1. Glomus tumours are common in NF1 and may be debilitating for affected patients.

Legius syndrome is characterized by the presence of multiple cafe-au-lait macules (CALMs), axillary freckling, macrocephaly and clinically overlaps with neurofibromatosis type 1 (NF1). However, Legius syndrome patients show a milder phenotype with tumoral complications systematically absent. Inactivating SPRED1 mutations were identified in Legius syndrome. SPRED1 interacts with neurofibromin and both proteins are negative regulators of the RAS-MAPK pathway. Constitutive Mismatch Repair Deficiency (CMMRD) is a differential diagnosis of NF1. CMMRD patients show in addition to multiple CALMs supplementary clinical features of NF1. Similar NF1 syndromes with CALMs are piebaldism, neurofibromatosis type 2, schwannomatosis, Noonan syndrome with multiple lentigines, McCune-Albright syndrome, and isolated cafe-au-lait spots. Differential diagnoses of NF1 are infantile myofibromatosis, Proteus syndrome, orbital neurofibromas, hyaline fibromatosis syndrome, and hybrid nerve sheath tumors.

The mechanical properties of the microenvironment have been increasingly accepted as potent modulators of cell behavior and function in physiological and pathological conditions. In tumors, tissue rigidity has been recognized as a potent driver of both neoplastic initiation from healthy tissue and progression toward malignancy. Several benign or malignant tumors are fibrotic and characterized by increased deposition, turnover, and posttranslational modifications of the extracellular matrix (ECM) that progressively stiffens the stroma. Likewise, neurofibromas are complex tumors composed of transformed Schwann cells, immersed in a heterogeneous fibrotic matrix. The molecular crosstalks that govern neurofibroma onset, growth, invasiveness, and progression toward malignancy remain poorly understood. In this chapter we propose that neurofibroma development requires the integration of potent pro-tumorigenic circuitries, such as those mastered by hyperactive Ras, and altered mechanosignaling axes conveyed by a stiff ECM, and that these pathways could be orchestrated by a pro-inflammatory extracellular milieu.

Due to the fact that NF1 is one of the more frequent genetic diseases (1/2500–1/3000, see Chap. 1), in the last 20 years dedicated centers for diagnosis and follow-up of these patients have been established allowing clinicians to acquire noteworthy clinical experience. Moreover, diagnostic laboratories are now able to provide molecular analyses with a detection rate that reaches 95% of patients affected by NF1. A critical revision of the classical grid of diagnostic criteria is now mandatory in order to actualize and mix the “classical” diagnostic criteria and recent knowledge. We remind that some of the proposed new clinical signs did not reach a unanimous consensus for different reasons among the experts to be introduced as new criteria, such as anemic nevus, UBOs, elevated head circumference and neuropsychological pattern.

The NF1 gene is a tumor suppressor which codes for neurofibromin. Biallelic NF1 inactivation has been identified in MPNSTs, Schwann cells from neurofibromas, glial cells of the peripheral nervous system, tumor cells of pheochromocytomas, gastro-intestinal stromal tumors, glomus cells from glomus tumors, tibial pseudarthrosis, and melanocytes from cafe-au-lait spots.

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.

Introduction/Background Recent research has distinguished various molecular classifications for endometrial carcinoma (EC). The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMise, Talhouk, BJC 2015,113:299) classification consists of immunohistochemistry (IHC) (p53 and mismatch repair proteins) and PCR sequencing for Polymerase Epsylon-Extracellular-Domain-Mutations (POLE-EDM). Methodology Ninety-three consecutive endometrial cancer patients, diagnosed between March 2017 and November 2018, were subjected to molecular and immunohistochemical analyses, according to ProMise. IHC for p53 and the mismatch repair proteins (MLH1, PMS2, MSH6 and PMS2) was performed. All patients were also tested for POLE-EDM using PCR, followed by Sanger sequencing (exon 9, 11, 13 and 14). In addition to the ProMise testing, all tumors and corresponding normal tissue of patients with mismatch repair deficient staining on IHC, were tested with PCR using the Microsatellite Instability (MSI) kit (MSI analysis system, Promega). Hypermethylation of MLH1 promotor and the presence of the BRAF p.V600E missense mutation was tested with (methylation specific) multiplex ligation dependent probe amplification. Results FIGO classification was stage IA(n=45), IB(n=17)) II(n=8), III(n=17) and IV(n=6). Classification according to histological type is presented in table 1. Of the 28 patients with MMR-D (Mismatch Repair-Deficiency) on IHC, 22(79%) showed hypermethylation as the probable cause of MMR-D. The remaining 6 patients were referred for germline analysis of Lynch syndrome. Five patients carried a pathogenic germline mutation in one of the mismatch repair genes: MSH6(n=3), PMS2(n=1), MLH1(n=1). In one patient no germline mutation was identified, hypothesizing a somatic cause. POLE-EDM was identified in 6 (6%) patients. Double positive status (POLE-EDM+MMR-D or POLE-EDM+p53 abnormal) was observed in 4 patients (4%) (table1). Conclusion The ProMise classification proved to be an efficient and easily implementable system. In the group with MMR-D, hypermethylation was the cause in 79%. POLE-EDM was identified in 6% of the patients, but its role needs to be clarified in non-endometrioid tumors. Disclosure Nothing to disclose.

Background Neurofibromatosis 1 (NF1) leads to the development of benign and malignant peripheral nerve sheath tumors (MPNST). MPNST have been described to develop in preexisting benign plexiform neurofibromas (PN) and have a poor prognosis. Atypical neurofibromas (ANF) were recently described as precursor lesions for MPNST, making early detection and management of ANF a possible strategy to prevent MPNST. We aimed to clinically characterize ANF and identify management approaches. Methods We analyzed clinical, imaging, and pathology findings of all patients with NF1 and ANF at 3 institutions. Results Sixty-three patients had 76 ANF (32M/31F; median age 27.1 y). On MRI, most ANF appeared as distinct nodular lesions and were 18F-fluorodeoxyglucose (FDG) avid. Forty-six ANF were associated with pain, 19 with motor weakness, 45 were palpable or visible, and 13 had no clinical signs. Completely resected ANF (N = 57) have not recurred (median follow-up, 4.1 y; range, 0-14 y). Four ANF transformed into MPNST and 17 patients had a history of MPNST in a different location than was their ANF. Conclusions Growth of distinct nodular lesions, pain, and FDG-PET avidity should raise concern for ANF in NF1. Patients with ANF are at greater risk for development of MPNST. Complete resection of ANF may prevent development of MPNST.

10530 Background: Germline pathogenic variants (PV) in the tumor suppressor gene TP53 are associated with a high risk of developing diverse malignancies, often at young age, and predispose to Li-Fraumeni syndrome (LFS). Surveillance programs for presymptomatic PV carriers have shown survival benefit in a non-randomized trial. Here we describe the surveillance findings and clinical outcomes of adults with TP53 PV undergoing a standardized screening protocol. Methods: We identified adults with germline PV in TP53 who underwent surveillance at the University Hospitals Leuven, Belgium, between 04/2013 and 08/2020. Patients with prior cancer were allowed, while patients with an active malignancy requiring treatment at diagnosis of the TP53 PV were excluded. Surveillance was performed per modified Toronto protocol, including annual whole body diffusion-weighted MRI (WB-DWI/MRI), brain MRI, abdominal ultrasound (US), endoscopic surveillance, laboratory tests, dermatological examination and breast MRI/US in females. The primary aim was to evaluate the number and type of malignancies and premalignant lesions diagnosed during screening and to assess the proportion of malignancies detected by surveillance. Secondary outcomes were the cancer detection rate during the first year of screening, the proportion of carriers with false-positive findings, and overall survival. Results: We included 42 adults from 20 apparently unrelated families. Median age was 38y (range, 17-70y) and 23 had a history of prior cancer. After a median follow-up of 41.5mo, we diagnosed 18 cancers in 12/42 participants (29%). Overall survival was 95% in all participants, including 2 carriers who opted to discontinue surveillance. Surveillance detected 10/18 cancers (56%), the majority of whom through WB-DWI/MRI (6/10; 60%). No malignancies were identified with brain MRI. In 5/42 individuals (12%), surveillance detected a malignancy during the first year of screening. Only 2/10 cancers discovered with surveillance (1 soft tissue and 1 bone sarcoma) belong to the LFS core tumors. Cancers not detected with surveillance (8/18) were 6 non-melanoma skin cancers and 2 interval cancers (sarcoma post radiation, secondary acute leukemia). Additionally, we detected 27 premalignant lesions in 11/42 patients (26%), of whom 78% were diagnosed by colonoscopy. False-positive findings occurred in 7/42 patients (17%) and were mostly seen with WB-DWI/MRI. Conclusions: Adults with germline PV in TP53 that undergo surveillance have high cancer detection rates. The majority of malignancies were asymptomatic at diagnosis and detected with WB-DWI/MRI. Despite the high cancer incidence, few LFS core cancers were diagnosed and survival was encouraging. Increased genetic testing changes the clinical picture of germline TP53 carrier populations, justifying the transition from LFS to a wider concept of heritable TP53-related cancer syndrome.

The Ras-MAPK and PI3K-AKT-mTOR signaling cascades were originally identified as cancer regulatory pathways but have now been demonstrated to be critical for synaptic plasticity and behavior. Neurodevelopmental disorders arising from mutations in these pathways exhibit related neurological phenotypes, including cognitive dysfunction, autism, and intellectual disability. The downstream targets of these pathways include regulation of transcription and protein synthesis. Other disorders that affect protein translation include fragile X syndrome (an important cause of syndromal autism), and other translational regulators are now also linked to autism. Here, we review how mechanisms of synaptic plasticity have been revealed by studies of mouse models for Ras-MAPK, PI3K-AKT-mTOR, and translation regulatory pathway disorders. We discuss the face validity of these mouse models and review current progress in clinical trials directed at ameliorating cognitive and behavioral symptoms.

The best known cafe-au-lait syndrome is neurofibromatosis type 1 (NF1). Legius syndrome (LS) is another, rarer syndrome with cafe-au-lait macules (CALMs). In young patients their clinical picture is often indistinguishable. We investigated the presence of choroidal abnormalities in syndromes with CALMs as a candidate tool for a more efficient diagnosis. Thirty-four patients with NF1 (14 with a truncating mutation, 14 with a non-truncating mutation and 6 with unknown mutation) and 11 patients with LS. All patients underwent an ophthalmological examination. Infrared images were performed. Choroidal nodules were diagnosed in 65% of the NF1 group. About 71% of NF1 patients with a truncating mutation and 50% of patients with a non-truncating mutation were found to have nodules. Choroidal nodules were seen in 18% of the LS patients, never more than one nodule/eye was detected in this group. Choroidal nodules are more abundantly present in NF1 genotypes with truncating mutations. In contrast, the number of choroidal nodules in LS is comparable with their presence in healthy individuals. Especially at an early age, when the clinical picture is incomplete, the detection of choroidal nodules is of diagnostic value, and helps in an appropriate genetic counselling and follow-up. These results support the suggestion to include choroidal nodules to the diagnostic criteria for NF1.

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.

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple cafe-au-lait macules, axillary freckling, learning disabilities and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the RAS-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids (aa) and the C-terminal 20 aa of the GTPase-Activating Protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120GAP. Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome. ispartof: Journal of Biological Chemistry vol:291 issue:7 pages:3124-34 ispartof: location:United States status: published