Identification of potential common genetic modifiers of neurofibromas: a genome-wide association study in 1333 patients with neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is characterized by the highly variable and unpredictable development of benign peripheral nerve sheath tumours: cutaneous (cNFs), subcutaneous (scNFs) and plexiform (pNFs) neurofibromas. OBJECTIVES: To identify neurofibroma modifier genes, in order to develop a database of patients with NF1. METHODS: All patients were phenotypically evaluated by a medical practitioner using a standardized questionnaire and the causal NF1 variant identified. We enrolled 1333 patients with NF1 who were genotyped for > 7 million common variants. RESULTS: A genome-wide association case-only study identified a significant association with 9q21.33 in the pNF phenotype in the discovery cohort. Twelve, three and four regions suggestive of association at the P ≤ 1 × 10-6 threshold were identified for pNFs, cNFs and scNFs, respectively. Evidence of replication was observed for 4, 2 and 6 loci, including 168 candidate modifier protein-coding genes. Among the candidate modifier genes, some were implicated in the RAS-mitogen-activated protein kinase pathway, cell-cycle control and myelination. Using an original CRISPR/Cas9-based functional assay, we confirmed GAS1 and SPRED2 as pNF and scNF candidate modifiers, as their inactivation specifically affected NF1-mutant Schwann cell growth. CONCLUSIONS: Our study may shed new light on the pathogenesis of NF1-associated neurofibromas and will, hopefully, contribute to the development of personalized care for patients with this deleterious and life-threatening condition.

Contribution of whole genome sequencing in the molecular diagnosis of mosaic partial deletion of the NF1 gene in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but highly variable expressivity. In most patients, Next Generation Sequencing (NGS) technologies allow the identification of a loss-of-function pathogenic variant in the NF1 gene, a negative regulator of the RAS-MAPK pathway. We describe the 5-year diagnosis wandering of a patient with a clear NF1 clinical diagnosis, but no molecular diagnosis using standard molecular technologies. The patient presented with a typical NF1 phenotype but NF1 targeted NGS, NF1 transcript analysis, MLPA, and array comparative genomic hybridization failed to reveal a genetic aberration. After 5 years of unsuccessful investigations, trio WGS finally identified a de novo mosaic (VAF ~ 14%) 24.6 kb germline deletion encompassing the promoter and first exon of NF1. This case report illustrates the relevance of WGS to detect structural variants including copy number variants that would be missed by alternative approaches. The identification of the causal pathogenic variant allowed a tailored genetic counseling with a targeted non-invasive prenatal diagnosis by detecting the deletion in plasmatic cell-free DNA from the proband's pregnant partner. This report clearly highlights the need to make WGS a clinically accessible test, offering a tremendous opportunity to identify a molecular diagnosis for otherwise unsolved cases.

Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis.

Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis. High EZH2 expression in tumors results from a tight coupling to proliferation to ensure H3K27me3 homeostasis. However, this process malfunctions in breast cancer. Low EZH2 expression relative to proliferation and mutations in Polycomb genes actually indicate poor prognosis and occur in metastases. We show that while altered EZH2 activity consistently modulates a subset of its target genes, it promotes a wider transcriptional instability. Importantly, transcriptional changes that are consequences of EZH2 loss are predominantly irreversible. Our study provides an unexpected understanding of EZH2's contribution to solid tumors with important therapeutic implications.

EZH1/2 function mostly within canonical PRC2 and exhibit proliferation-dependent redundancy that shapes mutational signatures in cancer.

Genetic mutations affecting chromatin modifiers are widespread in cancers. In malignant peripheral nerve sheath tumors (MPNSTs), Polycomb repressive complex 2 (PRC2), which plays a crucial role in gene silencing, is inactivated through recurrent mutations in core subunits embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), but mutations in PRC2's main catalytic subunit enhancer of zeste homolog 2 (EZH2) have never been found. This is in contrast to myeloid and lymphoid malignancies, which harbor frequent loss-of-function mutations in EZH2. Here, we investigated whether the absence of EZH2 mutations in MPNST is due to a PRC2-independent (i.e., noncanonical) function of the enzyme or to redundancy with EZH1. We show that, in the absence of SUZ12, EZH2 remains bound to EED but loses its interaction with all other core and accessory PRC2 subunits. Through genetic and pharmacological analyses, we unambiguously establish that EZH2 is functionally inert in this context, thereby excluding a PRC2-independent function. Instead, we show that EZH1 and EZH2 are functionally redundant in the slowly proliferating MPNST precursors. We provide evidence that the compensatory function of EZH1 is alleviated upon higher proliferation. This work reveals how context-dependent redundancies can shape tumor-type specific mutation patterns in chromatin regulators.

Accuracy of saliva and nasopharyngeal sampling for detection of SARS-CoV-2 in community screening: a multicentric cohort study.

Nasopharyngeal sampling for nucleic acid amplification testing (NAAT) is the standard diagnostic test of coronavirus disease 2019. Our objectives were to assess, in real-life conditions, the diagnostic accuracy of a nasopharyngeal point-of-care antigen (Ag) test and of saliva NAAT for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in ambulatory care. This was a prospective cohort study from 19 October through 18 December 2020 in two community COVID-19 screening centers in Paris, France. Two nasopharyngeal swabs and one saliva sample were simultaneously collected. Diagnostic accuracies of nasopharyngeal Ag testing and of three saliva NAAT methods were assessed as compared to nasopharyngeal NAAT. A total of 1452 ambulatory children and adults were included. Overall, 129/1443 (9%) participants tested positive on nasopharyngeal NAAT (102/564 [18%] in symptomatic and 27/879 [3%] in asymptomatic participants). Sensitivity was 94%, 23%, 96%, and 94% for the three different protocols of saliva NAAT and for the nasopharyngeal Ag test, respectively. Estimates of specificity were above 95% for all methods. Diagnostic accuracy was similar in symptomatic and asymptomatic individuals. Diagnostic accuracy of nasopharyngeal Ag testing and of saliva NAAT is similar to that of nasopharyngeal NAAT, subject to compliance with specific protocols for saliva. Registration number: NCT04578509.

Chemoresistant pleomorphic rhabdomyosarcoma: whole exome sequencing reveals underlying cancer predisposition and therapeutic options.

BACKGROUND: Rhabdomyosarcoma (RMS) is rare cancer affecting children and adults. Pleomorphic RMS histology is almost exclusive to adult patients and often resistant to chemotherapy. OBJECTIVE: We report the case of a 19-year-old patient who presented with a metastatic chemoresistant pleomorphic RMS. METHODS: Considering the poor prognosis and the few systemic therapeutic options, we decided to carry out a whole exome sequencing (WES) of the tumour and germline DNA. RESULTS: WES identified a germline variation (c.1863_1864insT) in the MLH1 gene corresponding to a pathogenic mutation: (p. Leu622Serfs*10), whereas the family history did not fit with classical criteria for Lynch syndrome. Loss-of-heterozygosity at MLH1 locus was found in the tumour. Immunohistochemistry showed loss of MLH1 and PMS2 nuclear expression in the tumour cells. In view of the mismatch repair defects and a high programmed cell death ligand 1 (PD-L1) expression (60% of tumour cells expressed PD-L1), we administrated an anti-PD-1 antibody to the patient. He achieved a rapid complete response of the lung metastases, which appears sustained after a 1-year follow-up. CONCLUSION: This observation of an RMS revealing an unexpected Lynch syndrome underlines the overlap between tumorous and germline molecular genetics and emphasises the major impact of cancer genomic medicine in clinical practice for guiding treatment decision.

PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies.

The polycomb repressive complex 2 (PRC2) exerts oncogenic effects in many tumour types. However, loss-of-function mutations in PRC2 components occur in a subset of haematopoietic malignancies, suggesting that this complex plays a dichotomous and poorly understood role in cancer. Here we provide genomic, cellular, and mouse modelling data demonstrating that the polycomb group gene SUZ12 functions as tumour suppressor in PNS tumours, high-grade gliomas and melanomas by cooperating with mutations in NF1. NF1 encodes a Ras GTPase-activating protein (RasGAP) and its loss drives cancer by activating Ras. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly, however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies not only reveal an unexpected connection between the PRC2 complex, NF1 and Ras, but also identify a promising epigenetic-based therapeutic strategy that may be exploited for a variety of cancers.

Non-invasive prenatal diagnosis of paternally inherited disorders from maternal plasma: detection of NF1 and CFTR mutations using droplet digital PCR.

BACKGROUND: To limit risks of miscarriages associated with invasive procedures of current prenatal diagnosis practice, we aim to develop a personalized medicine-based protocol for non-invasive prenatal diagnosis (NIPD) of monogenic disorders relying on the detection of paternally inherited mutations in maternal blood using droplet digital PCR (ddPCR). METHODS: This study included four couples at risk of transmitting paternal neurofibromatosis type 1 (NF1) mutations and four couples at risk of transmitting compound heterozygous CFTR mutations. NIPD was performed between 8 and 15 weeks of gestation, in parallel to conventional invasive diagnosis. We designed specific hydrolysis probes to detect the paternal mutation and to assess the presence of cell-free fetal DNA by ddPCR. Analytical performances of each assay were determined from paternal sample, an then fetal genotype was inferred from maternal plasma sample. RESULTS: Presence or absence of the paternal mutant allele was correctly determined in all the studied plasma DNA samples. CONCLUSIONS: We report an NIPD protocol suitable for implementation in an experienced laboratory of molecular genetics. Our proof-of-principle results point out a high accuracy for early detection of paternal NF1 and CFTR mutations in cell-free DNA, and open new perspectives for extending the technology to NIPD of many other monogenic diseases.

CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder caused by the accumulation of very long-chain fatty acids (VLCFA) due to mutations in the ABCD1 gene. The phenotypic spectrum ranges from a fatal cerebral demyelinating disease in childhood (cerebral ALD) to a progressive myelopathy without cerebral involvement in adulthood (adrenomyeloneuropathy). Because ABCD1 mutations have no predictive value with respect to clinical outcome a role for modifier genes was postulated. We report that the CYP4F2 polymorphism rs2108622 increases the risk of developing cerebral ALD in Caucasian patients. The rs2108622 polymorphism (c.1297G>A) results in an amino acid substitution valine for methionine at position 433 (p.V433M). Using cellular models of VLCFA accumulation, we show that p.V433M decreases the conversion of VLCFA into very long-chain dicarboxylic acids by ω-oxidation, a potential escape route for the deficient peroxisomal ß-oxidation of VLCFA in ALD. Although p.V433M does not affect the catalytic activity of CYP4F2 it reduces CYP4F2 protein levels markedly. These findings open perspectives for therapeutic interventions in a disease with currently limited treatment options.

SETD2 and DNMT3A screen in the Sotos-like syndrome French cohort.

BACKGROUND: Heterozygous NSD1 mutations were identified in 60%-90% of patients with Sotos syndrome. Recently, mutations of the SETD2 and DNMT3A genes were identified in patients exhibiting only some Sotos syndrome features. Both NSD1 and SETD2 genes encode epigenetic 'writer' proteins that catalyse methylation of histone 3 lysine 36 (H3K36me). The DNMT3A gene encodes an epigenetic 'reader' protein of the H3K36me chromatin mark. METHODS: We aimed at confirming the implication of DNMT3A and SETD2 mutations in an overgrowth phenotype, through a comprehensive targeted-next generation sequencing (NGS) screening in 210 well-phenotyped index cases with a Sotos-like phenotype and no NSD1 mutation, from a French cohort. RESULTS: Six unreported heterozygous likely pathogenic variants in DNMT3A were identified in seven patients: two nonsense variants and four de novo missense variants. One de novo unreported heterozygous frameshift variant was identified in SETD2 in one patient. All the four DNMT3A missense variants affected DNMT3A functional domains, suggesting a potential deleterious impact. DNMT3A-mutated index cases shared similar clinical features including overgrowth phenotype characterised by postnatal tall stature (≥+2SD), macrocephaly (≥+2SD), overweight or obesity at older age, intellectual deficiency and minor facial features. The phenotype associated with SETD2 mutations remains to be described more precisely. The p.Arg882Cys missense de novo constitutional DNMT3A variant found in two patients is the most frequent DNMT3A somatic mutation in acute leukaemia. CONCLUSIONS: Our results illustrate the power of targeted NGS to identify rare disease-causing variants. These observations provided evidence for a unifying mechanism (disruption of apposition and reading of the epigenetic chromatin mark H3K36me) that causes an overgrowth syndrome phenotype. Further studies are needed in order to assess the role of SETD2 and DNMT3A in intellectual deficiency without overgrowth.

Identification by FFPE RNA-Seq of a new recurrent inversion leading to RBM10-TFE3 fusion in renal cell carcinoma with subtle TFE3 break-apart FISH pattern.

Gene fusions involving TFE3 defines the "Xp11.2 translocations" subclass of renal cell carcinomas (RCCs) belonging to the MiT family translocation RCC. Four recurrent TFE3 fusion partners were identified to date: PRCC, ASPSCR1, SFPQ, and NONO. Break-apart TFE3 fluorescence in situ hybridization (FISH) on formalin-fixed and paraffin-embedded (FFPE) tissue sections is currently the gold standard for identification of TFE3 rearrangements. Herein, we report a case of RCC with a morphological appearance of Xp11.2 translocation, and positive TFE3 immunostaining. By FISH, the spots constituting the split signal were barely spaced, suggestive of a chromosome X inversion rather than a translocation. We performed RNA-seq from FFPE material to test this hypothesis. RNA-seq suggested a fusion of RBM10 gene exon 17 (Xp11.23) with TFE3 gene exon 5 (Xp11.2). RBM10-TFE3 fusion transcript was confirmed using specific RT-PCR. Our work showed that RNA-Seq is a robust technique to detect fusion transcripts from FFPE material. A RBM10-TFE3 fusion was previously described in single case of Xp11.2 RCC. Although rare, RBM10-TFE3 fusion variant (from chromosome X paracentric inversion), therefore, appears to be a recurrent molecular event in Xp11.2 RCCs. RBM10-TFE3 fusion should be added in the list of screened fusion transcripts in targeted molecular diagnostic multiplex RT-PCR. © 2016 Wiley Periodicals, Inc.

CYP46A1 inhibition, brain cholesterol accumulation and neurodegeneration pave the way for Alzheimer's disease.

Abnormalities in neuronal cholesterol homeostasis have been suspected or observed in several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and Huntington's disease. However, it has not been demonstrated whether an increased abundance of cholesterol in neurons in vivo contributes to neurodegeneration. To address this issue, we used RNA interference methodology to inhibit the expression of cholesterol 24-hydroxylase, encoded by the Cyp46a1 gene, in the hippocampus of normal mice. Cholesterol 24-hydroxylase controls cholesterol efflux from the brain and thereby plays a major role in regulating brain cholesterol homeostasis. We used an adeno-associated virus vector encoding short hairpin RNA directed against the mouse Cyp46a1 mRNA to decrease the expression of the Cyp46a1 gene in hippocampal neurons of normal mice. This increased the cholesterol concentration in neurons, followed by cognitive deficits and hippocampal atrophy due to apoptotic neuronal death. Prior to neuronal death, the recruitment of the amyloid protein precursor to lipid rafts was enhanced leading to the production of ß-C-terminal fragment and amyloid-ß peptides. Abnormal phosphorylation of tau and endoplasmic reticulum stress were also observed. In the APP23 mouse model of Alzheimer's disease, the abundance of amyloid-ß peptides increased following inhibition of Cyp46a1 expression, and neuronal death was more widespread than in normal mice. Altogether, these results suggest that increased amounts of neuronal cholesterol within the brain may contribute to inducing and/or aggravating Alzheimer's disease.

Reduction of microRNA 122 expression in IFNL3 CT/TT carriers and during progression of fibrosis in patients with chronic hepatitis C.

UNLABELLED: The microRNA miR-122 is highly expressed in the liver and stimulates hepatitis C virus (HCV) replication in vitro. IFNL3 (lambda-3 interferon gene) polymorphisms and the expression of miR-122 have been associated with sustained virological response (SVR) to treatment with pegylated interferon plus ribavirin in patients with chronic hepatitis C (CHC). We investigated, in vivo, the relationship between miR-122 expression, IFNL3 polymorphism, fibrosis, and response to PEG-IFN plus ribavirin. Pretreatment liver biopsy specimens and serum samples from 133 patients with CHC were included. Sixty-six patients achieved SVR, and 64 failed to respond to the treatment (43 nonresponders [NR] and 21 relapsers [RR]). All stages of fibrosis were represented, with 39, 50, 23, and 19 patients, respectively, having Metavir scores of F1, F2, F3, and F4. miR-122 expression was assessed by real-time quantitative PCR (RT-qPCR) and IFNL3 rs12979860 by direct sequencing. Hepatic miR-122 expression was higher in patients with the IFNL3 CC genotype than in those with the IFNL3 CT or TT genotype, in all patients (P = 0.025), and in NRs plus RRs (P = 0.013). Increased hepatic miR-122 was more strongly associated with complete early virological response (cEVR) (P = 0.003) than with SVR (P = 0.016). In multivariate analysis, increased hepatic miR-122 was only associated with the IFNL3 CC genotype. miR-122 was decreased in patients with advanced fibrosis (Metavir scores of F3 and F4) compared to its levels in patients with mild and moderate fibrosis (F1 and F2) (P = 0.01). Serum and hepatic expression of miR-122 were not associated. The association between miR-122 and IFNL3 was stronger than the association between miR-122 and response to treatment. miR-122 may play a role in the early viral decline that is dependent on IFNL3 and the innate immune response. IMPORTANCE: miR-122 plays a crucial role during HCV infection. Moreover, it was reported that miR-122 binding within the HCV genome stimulates its replication. Moreover, miR-122 is highly expressed within hepatocytes, where it regulates many cellular pathways. A reduction of miR-122 expression has been suggested to be associated with responsiveness to IFN-based therapy in patients with chronic hepatitis C. Several independent genome-wide association studies reported a strong association between IFNL3 polymorphism and responsiveness to IFN-based therapy. We report here a strong association between the expression of miR-122 and IFNL3 polymorphism that is independent of the response to the treatment. Our data suggest that modification of miR-122 expression may play an important role in the molecular mechanism associated with IFNL3 polymorphism. Moreover, we report a reduction of miR-122 at more advanced stages of fibrosis in patients with chronic hepatitis C.

NF1 single and multi-exons copy number variations in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is caused by dominant loss-of-function mutations of the tumor suppressor NF1 containing 57 constitutive coding exons. A huge number of different pathogenic NF1 alterations has been reported. The aim of the present study was to evaluate the usefulness of a multiplex ligation-dependent probe amplification (MLPA) approach in NF1 patients to detect single and multi-exon NF1 gene copy number variations. A genotype-phenotype correlation was then performed in NF1 patients carrying these types of genetic alterations. Among 565 NF1 index cases from the French NF1 cohort, single and multi-exon deletions/duplications screening identified NF1 partial deletions/duplications in 22 patients (~4%) using MLPA analysis. Eight single exon deletions, 11 multiple exons deletions, 1 complex rearrangement and 2 duplications were identified. All results were confirmed using a custom array-CGH. MLPA and custom array-CGH allowed the identification of rearrangements that were missed by cDNA/DNA sequencing or microsatellite analysis. We then performed a targeted next-generation sequencing of NF1 that allowed confirmation of all 22 rearrangements. No clear genotype-phenotype correlations were found for the most clinically significant disease features of NF1 in patients with single and multi-exons NF1 gene copy number changes.

Mutations in SETD2 cause a novel overgrowth condition.

BACKGROUND: Overgrowth conditions are a heterogeneous group of disorders characterised by increased growth and variable features, including macrocephaly, distinctive facial appearance and various degrees of learning difficulties and intellectual disability. Among them, Sotos and Weaver syndromes are clinically well defined and due to heterozygous mutations in NSD1 and EZH2, respectively. NSD1 and EZH2 are both histone-modifying enzymes. These two epigenetic writers catalyse two specific post-translational modifications of histones: methylation of histone 3 lysine 36 (H3K36) and lysine 27 (H3K27). We postulated that mutations in writers of these two chromatin marks could cause overgrowth conditions, resembling Sotos or Weaver syndromes, in patients with no NSD1 or EZH2 abnormalities. METHODS: We analysed the coding sequences of 14 H3K27 methylation-related genes and eight H3K36 methylation-related genes using a targeted next-generation sequencing approach in three Sotos, 11 'Sotos-like' and two Weaver syndrome patients. RESULTS: We identified two heterozygous mutations in the SETD2 gene in two patients with 'Sotos-like' syndrome: one missense p.Leu1815Trp de novo mutation in a boy and one nonsense p.Gln274* mutation in an adopted girl. SETD2 is non-redundantly responsible for H3K36 trimethylation. The two probands shared similar clinical features, including postnatal overgrowth, macrocephaly, obesity, speech delay and advanced carpal ossification. CONCLUSIONS: Our results illustrate the power of targeted next-generation sequencing to identify rare disease-causing variants. We provide a compelling argument for Sotos and Sotos-like syndromes as epigenetic diseases caused by loss-of-function mutations of epigenetic writers of the H3K36 histone mark.

Prostate cancer outcomes in France: treatments, adverse effects and two-year mortality.

BACKGROUND: This very large population-based study investigated outcomes after a diagnosis of prostate cancer (PCa) in terms of mortality rates, treatments and adverse effects. METHODS: Among the 11 million men aged 40 years and over covered by the general national health insurance scheme, those with newly managed PCa in 2009 were followed for two years based on data from the national health insurance information system (SNIIRAM). Patients were identified using hospitalisation diagnoses and specific refunds related to PCa and PCa treatments. Adverse effects of PCa treatments were identified by using hospital diagnoses, specific procedures and drug refunds. RESULTS: The age-standardised two-year all-cause mortality rate among the 43,460 men included in the study was 8.4%, twice that of all men aged 40 years and over. Among the 36,734 two-year survivors, 38% had undergone prostatectomy, 36% had been treated by hormone therapy, 29% by radiotherapy, 3% by brachytherapy and 20% were not treated. The frequency of treatment-related adverse effects varied according to age and type of treatment. Among men between 50 and 69 years of age treated by prostatectomy alone, 61% were treated for erectile dysfunction and 24% were treated for urinary disorders. The frequency of treatment for these disorders decreased during the second year compared to the first year (erectile dysfunction: 41% vs 53%, urinary disorders: 9% vs 20%). The frequencies of these treatments among men treated by external beam radiotherapy alone were 7% and 14%, respectively. Among men between 50 and 69 years with treated PCa, 46% received treatments for erectile dysfunction and 22% for urinary disorders. For controls without PCa but treated surgically for benign prostatic hyperplasia, these frequencies were 1.5% and 6.0%, respectively. CONCLUSIONS: We report high survival rates two years after a diagnosis of PCa, but a high frequency of PCa treatment-related adverse effects. These frequencies remain underestimated, as they are based on treatments for erectile dysfunction and urinary disorders and do not reflect all functional outcomes. These results should help urologists and general practitioners to inform their patients about outcomes at the time of screening and diagnosis, and especially about potential treatment-related adverse effects.

Droplet Digital PCR for Fast and Accurate Characterization of NF1 Locus Deletions: Confirmation of the Predominant Maternal Origin of Type-1 Deletions.

Neurofibromatosis type-1 is a genetic disorder caused by loss-of-function variants in the tumor-suppressor NF1. Approximately 4% to 11% of neurofibromatosis type-1 patients have a NF1 locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype-phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) set along the NF1 locus was designed to delimitate the three recurrent NF1 deletion breakpoints. The ddPCR was tested in 121 samples from nonrelated NF1-deleted patients. Classification based on ddPCR versus multiplex ligation-dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type-1 (64%), 20 type-2 (16%), 7 type-3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type-2 using MLPA, for which the SUZ12 gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. This study proposes a fast and efficient ddPCR quantification to allow fine NF1 deletion classification. It indicates that ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1 point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling.

NF1 molecular characterization and neurofibromatosis type I genotype-phenotype correlation: the French experience.

Neurofibromatosis type 1 (NF1) affects about one in 3,500 people in all ethnic groups. Most NF1 patients have private loss-of-function mutations scattered along the NF1 gene. Here, we present an original NF1 investigation strategy and report a comprehensive mutation analysis of 565 unrelated patients from the NF-France Network. A NF1 mutation was identified in 546 of the 565 patients, giving a mutation detection rate of 97%. The combined cDNA/DNA approach showed that a significant proportion of NF1 missense mutations (30%) were deleterious by affecting pre-mRNA splicing. Multiplex ligation-dependent probe amplification allowed the identification of restricted rearrangements that would have been missed if only sequencing or microsatellite analysis had been performed. In four unrelated families, we identified two distinct NF1 mutations within the same family. This fortuitous association points out the need to perform an exhaustive NF1 screening in the case of molecular discordant-related patients. A genotype-phenotype study was performed in patients harboring a truncating (N = 368), in-frame splicing (N = 36), or missense (N = 35) mutation. The association analysis of these mutation types with 12 common NF1 clinical features confirmed a weak contribution of the allelic heterogeneity of the NF1 mutation to the NF1 variable expressivity.

MicroRNAome profiling in benign and malignant neurofibromatosis type 1-associated nerve sheath tumors: evidences of PTEN pathway alterations in early NF1 tumorigenesis.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a common dominant tumor predisposition syndrome affecting 1 in 3,500 individuals. The hallmarks of NF1 are the development of peripheral nerve sheath tumors either benign (dermal and plexiform neurofibromas) or malignant (MPNSTs). RESULTS: To comprehensively characterize the role of microRNAs in NF1 tumorigenesis, we analyzed 377 miRNAs expression in a large panel of dermal and plexiform neurofibromas, and MPNSTs. The most significantly upregulated miRNA in plexiform neurofibromas was miR-486-3p that targets the major tumor suppressor gene, PTEN. We confirmed PTEN downregulation at mRNA level. In plexiform neurofibromas, we also report aberrant expression of four miRNAs involved in the RAS-MAPK pathway (miR-370, miR-143, miR-181a, and miR-145). In MPNSTs, significant deregulated miRNAs were involved in PTEN repression (miR-301a, miR-19a, and miR-106b), RAS-MAPK pathway regulation (Let-7b, miR-195, and miR-10b), mesenchymal transition (miR-200c, let-7b, miR-135a, miR-135b, and miR-9), HOX genes expression (miR-210, miR-196b, miR-10a, miR-10b, and miR-9), and cell cycle progression (miR-195, let-7b, miR-20a, miR-210, miR-129-3p, miR-449a, and miR-106b). CONCLUSION: We confirmed the implication of PTEN in genesis of plexiform neurofibromas and MPNSTs in NF1. Markedly deregulated miRNAs might have potential diagnostic or prognostic value and could represent novel strategies for effective pharmacological therapies of NF1 tumors.

Neurofibromatosis type 1: from genotype to phenotype.

Although neurofibromatosis 1 (NF1) is a common Mendelian disorder with autosomal-dominant inheritance, its expression is highly variable and unpredictable. Many NF1 patients have been genotyped but few allele-phenotype correlations have been identified. NF1 genotype-phenotype correlations are difficult to identify because of the complexity of the NF1 phenotype, its strong age dependency, the relatedness of many clinical features and the huge heterogeneity of pathogenic NF1 mutations. Some NF1 patients with a given NF1 mutation may develop very severe disease while others with the same mutation have only mild symptoms. This phenotypic variability may be due to both modifier genes and environmental factors. Recent targeted strategies have identified several interesting candidate modifier genes.