Neurofibromatosis Type 1 Families Research Articles

Neuroblastic tumors and neurofibromatosis type 1: A retrospective multicenter study in Italy and systematic review of the literature.

Neuroblastic tumors (NBTs) are the most common extra-cranial solid tumors of childhood. Neurofibromatosis type 1 (NF1) is the most common neurocutaneous disorder with a predisposition to tumors. The co-occurrence of NBTs in the setting of NF1 has been occasionally reported, suggesting a non-casual association and likely configuring a spectrum of neural crest-derived disorders. To explore the occurrence of NBTs within NF1 and to report on its natural history, therapeutic strategies, and outcomes in an Italian cohort of children with NF1 and in the literature. Study (a): a retrospective analysis of questionnaire-based data [years 1979-2017] derived from the databases of the Italian Registry for Neuroblastoma (RINB) of the Italian Society of Pediatric Onco-Haematology (AIEOP); and Study (b): a systematic review search on NF1/NB co-occurrence. Study (a) identified eight children with NBTs, 0.2% of patients registered in the RINB, fulfilling the diagnostic criteria for NF1. The primary site of NBTs was abdominal in six patients. The NBTs were neuroblastoma (NB) in five patients, ganglioneuroblastoma (GNB) in one, patient, and ganglioneuroma (GN) in two. Metastatic diffusion occurred in three out of eight children. MYCN gene testing, performed in the tumors of five patients, resulted not-amplified. The major features of NF1 included the following: NF1 family history in four patients, café-au-lait spots in all, freckling in six, Lisch nodules in three, and neurofibromas in three. With regard to the outcome, four children survived three of these for the progression of NB and one for a second tumor. Study (b) identified 12 patients with NF1/NB from the years 1966-2017, and the median age at diagnosis was 27 months (range = 0-168 months). The primary site of NB was thoracic. The prevalent histotype was NB in nine patients, GNB in two, and GN in one. Eight/nine NBs were metastatic. The MYCN gene was amplified in the only studied case. The NF1 features included NF1 family history in seven patients; the major NF1 features were café-au-lait spots in nine patients, freckling in one, Lisch nodules in none, and neurofibromas in six. The outcome was good for only two children, while eight children died of neuroblastoma, at a median age of 49.5 months (range = 2.4-174 months), with a median survival time of 21.75 months after diagnosis. To our knowledge, this represents the first systematic study on the occurrence of NBTs in NF1. This confirms that NBs are rare per se in the setting of NF1 (0.2% of all NBs) and even if compared to the overall frequency of malignancies in NF1 (i.e., 14.7%). The male:female ratio in study (a) (0.6) was different from what was recorded in study (b) (1.5) and in line with the overall increased frequency of malignancies in females with NF1. The median ages at diagnosis of NB in either study (a) or (b) were concordant with what occurred in the NB population. In study (a) versus study (b), the frequency of metastatic diffusion was lower, likely indicating less awareness on work-ups for malignancies in old NF1 series in the literature. The outcome was much better in study (a) than in study (b), indicating that multidisciplinary treatment for NB is highly recommended.

Prospects for the treatment of neurofibromatosis type 1: A review

Neurofibromatosis type 1 (NF1) is an autosomal dominant hereditary tumor syndrome with a prevalence of 1:3000 in human population. About 50% of NF1 cases are sporadic due to newly emerging germline mutations in NF1 gene. Protein product of NF1 is a neurofibromin, which inhibits RAS-RAF-MEK-ERK system. The prevalence of NF1 is increasing as patients are fertile. Therefore, it is important to use rapid diagnostic methods for NF1 mutations in NF1 families for prenatal prophylaxis. Mutations in NF1 gene play roles in sporadic carcinogenesis and in development of cancer resistance to chemotherapy. Specific for NF1 are multiple subcutaneous and cutaneous neurofibromas, age spots, skeletal abnormalities, mental retardation, tumors of the brain and optic nerves. Half of patients with NF1 develop plexiform neurofibromas, which disfigure them or compress vital organs. The difficulty in treating NF1 is due to involvement of immune system, since a large number of degranulating mast cells are found in neurofibromas. Mast cells secrete cytokines that dont provide a proper anti-tumor immune response, but initiate formation and growth of new neurofibromas. Therefore, long-term administration of ketotiphen was proposed for treatment of NF1 patients. Surgical removal of neurofibromas causes relapses and induction of the growth of new tumors; therefore, it is necessary to develop an effective therapy for NF1. The effectiveness of complex therapy of NF1 with use of ketotiphen, Lydase and Aevit, as well as monotherapy with an ATP-independent inhibitor of mitogen-activated protein kinase, has been described. For widespread clinical implementation of these methods, it is necessary to conduct studies on large sample of patients, as well as to make medicines available for patients. Gene therapy may become promising in the treatment of NF1, which requires identification of the type of mutation in NF1 gene in each individual and the use of specific microRNAs.

Clinical presentation and genetic analyses of neurofibromatosis type 1 in independent patients with monoallelic double de novo closely spaced mutations in the NF1 gene.

Neurofibromatosis type 1 (NF1) belongs to RASopathies, a group of syndromes caused by germline mutations in Ras/MAPK pathway genes. Most NF1 patients exhibit single inactivating pathogenic variants within the NF1 gene. We performed extensive genetic analyses in two NF1 families disclosing the first two cases of double de novo monoallelic NF1 variants. Both index patients described in this study had classical NF1. Probands were born from fathers in their late 30s and presented closely spaced double mutations (<100 bp) in NF1 regions showing an excess of somatic mutations. Closely spaced multiple mutations have been reported in RAS/MAPK signaling genes but never in NF1. Mutagenesis is a quasi‐random process in humans, therefore two causative variants in the same gene, moreover in the same allele are exceptional. Here, we discuss possible mechanisms for this ultrarare event. Our findings confirm the possibility of a higher risk of concurrent de novo variants in NF1.

Neurofibromatosis type 1 families with first-degree relatives harbouring distinct NF1 pathogenic variants. Genetic counselling and familial diagnosis: what should be offered?

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by pathogenic variants in NF1. Recently, NF1 testing has been included as a clinical criterion for NF1 diagnosis. Additionally, preconception...

Identification of a Novel NF1 Frameshift Variant in a Chinese Family with Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a progressive neurocutaneous disorder in humans, mainly characterized by café-au-lait macules (CALMs) and neurofibromas. NF1 is caused by variants of the neurofibromin 1 gene (NF1), which encodes a Ras-GTPase-activating protein called neurofibromin. NF1 variants may result in loss of neurofibromin function and elevation of cell proliferation and tumor formation. In this study, a Chinese NF1 family with an autosomal dominant inheritance pattern was recruited. Exome sequencing and Sanger sequencing were performed to discover the causative variant responsible for the family, followed by molecular analysis of effect of the mutated NF1 protein on Ras activity. A novel frameshift variant c.541dupC (p.(Gln181Profs∗20)) in the NF1 gene was identified in all three affected family members. The variant cosegregated with the disease phenotypes in the pedigree and was absent in 100 healthy controls. Bioinformatic analysis showed that the variant c.541dupC (p.(Gln181Profs∗20)) was pathogenic. The further molecular analysis verified the cells expressing NF1 variant p.(Gln181Profs∗20) partially enhanced Ras activity and elevated cell proliferation and tumor formation due to loss of neurofibromin function caused by the variant. Taken together, the data strongly advocate the c.541dupC (p.(Gln181Profs∗20)) variant as the underlying genetic cause of the Chinese family with NF1. Moreover, our findings broaden the spectrum of NF1 variants and provide molecular insights into the pathogenesis of NF1.

A Novel Deep Intronic Mutation Introducing a Cryptic Exon Causing Neurofibromatosis Type 1 in a Family with Highly Variable Phenotypes: A Case Study

Neurofibromatosis type 1 (NF1) is a common dominant inherited disorder with highly variable expressivity. Genetic testing for this condition has been more available the last decade. Here we present a case report of a NF1 family including seven affected family members, some of them with a very severe phenotype. When searching for a causative mutation in the NF1 gene, no mutation was found at DNA level. However, a misspliced transcript including a subsequence of intron 3 appeared when screening RNA. The underlying cause at DNA level was determined to be a deep intronic variant (c.288+1137C>T). This intronic point mutation creates a new splice site causing the insertion of a cryptic exon (r.288_289ins288+1018_1135), leading to reading frameshift at the protein level. Deep intronic mutations introducing a cryptic exon are known to be a cause of NF1, and we reviewed the literature to evaluate how common this mutation is in NF1 syndrome. We found 20 different deep intronic NF1 splice mutations, including the one found in the present study. In conclusion, this case illustrates the value of RNA analysis to detect the cause of genetic diseases, and we decided to use RNA based mutation screening as standard procedure for NF1 genetic testing in our laboratory.

Identification of the NF1 gene mutation in Korean families with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1), the autosomal dominant diseases in humans, is caused by defects in the NF1 tumor-suppressor gene. To date, more than 700 independent mutations have been identified in the NF1 gene, the majority of which are family specific. The aim of this study was to investigate the pathogenic mutations in NF1 gene in 15 Korean NF1 families. We performed genetic analysis on 37 patients from 15 unrelated families by using RT-PCR and mRNA sequencing with 12 primer set including whole coding region of the NF1 gene. Here, we identified the familial and sporadic mutations in 34 of 37 patients (91.9 %) and found the familial mutations in 11 of 15 families (73.3 %). We detected 29 mutations of NF1 gene, which may encode the truncated form of neurofibromin. Four mutations were found in the cAMP-dependent protein kinase recognition sites (G1610A, c.2179delCAG) and the GTPase activating protein related domain (c.3169insT, c.2863delC). In this series of familial studies, we report a wide spectrum of NF1 mutations in Korean patients. We conclude that most mutations caused abnormal transcripts and premature termination of the mutant alleles. Then we suggest that these may provide further insights into the pathogenesis of NF1.

Clinico-pathological and biomolecular findings in Italian patients with multiple cutaneous neurofibromas

BackgroundNeurofibroma occurs as isolated or multiple lesions frequently associated with neurofibromatosis type 1 (NF1), a common autosomal dominant disorder affecting 1 in 3500 individuals. It is caused by mutations in the NF1 gene, which comprises 60 exons and is located on chromosome 17q11.2. NF1 is a fully penetrant gene exhibiting a mutation rate some 10-fold higher compared with most other disease genes. As a consequence, a high number of cases (up to 50%) are sporadic. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of lesions.Methods110 patients with at least two neurofibroma lesions recorded in the files of the Pathology Department of the University of Modena during the period 1999-2010, were included in this study. Through interviews and examination of clinical charts, pedigrees were drawn for all patients who were affected by at least two neurofibromas. We attempted to delineate the clinical features of NF1 and the mutational spectrum in the cohort of 11 NF1 families identified. For each proband, the whole coding sequence and all splice sites were studied for mutations, either by the protein truncation test (PTT), or, more frequently, by denaturing high performance liquid chromatography (DHPLC). Two GIST tumors of NF1 patients were tested for somatic NF1 mutations.ResultsNF1 germline mutations were identified in 7 (68%) patients. A novel mutation, c.3457_3460delCTCA in exon 20, was detected in two unrelated patients and was associated with different clinical features. No NF1 somatic mutations were detected in the GIST tumors. A wide phenotypic and genotypic variability was registered, both in the spectrum of skin lesions and visceral neoplasms, even among members of the same family who had different clinical manifestations. A proclivity to multiple tumors arising in the same subject, and a higher tumor burden per family were the most relevant findings observed in patients affected with the NF1 mutation.ConclusionsWe report a novel NF1 mutation and we contribute data for the refinement of the NF1 genotype-phenotype spectrum.

SPRED1 mutations (Legius syndrome): another clinically useful genotype for dissecting the neurofibromatosis type 1 phenotype

Objective:Mutations of the SPRED1 gene, one of a family of Sprouty (Spry)/Spred proteins known to “downregulate” mitogen activated protein kinase (MAPK) signalling, have been identified in patients with a mild...

Unravelling the genetic basis of variable clinical expression in neurofibromatosis 1

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder which displays considerable inter- and intra-familial variability in phenotypic expression. To evaluate the genetic component of variable expressivity in NF1, we examined the phenotypic correlations between affected relatives in 750 NF1 patients from 275 multiplex families collected through the NF-France Network. Twelve NF1-related clinical features, including five quantitative traits (number of café-au-lait spots of small size and of large size, and number of cutaneous, subcutaneous and plexiform neurofibromas) and seven binary ones, were scored. All clinical features studied, with the exception of neoplasms, showed significant familial aggregation after adjusting for age and sex. For most of them, patterns of familial correlations indicated a strong genetic component with no apparent influence of the constitutional NF1 mutation. Heritability estimates of the five quantitative traits ranged from 0.26 to 0.62. Moreover, we investigated for the first time the role of the normal NF1 allele in the variable expression of NF1 through a family-based association study. Nine tag SNPs in NF1 were genotyped in 1132 individuals from 313 NF1 families. No significant deviations of transmission of any of the NF1 variants to affected offspring was found for any of the 12 clinical features examined, based on single marker or haplotype analysis. Taken together, our results provided evidence that genetic modifiers, unlinked to the NF1 locus, contribute to the variable expressivity of the disease.

Nf1 expression is dependent on strain background: implications for tumor suppressor haploinsufficiency studies

Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with elevated risk for both astrocytoma and peripheral nerve sheath tumors. NF1 is caused by a germline mutation in the NF1 gene, with tumors showing loss of the wild type copy of NF1. In addition, NF1 heterozygosity in surrounding stroma is important for tumor formation, suggesting an additional role of haploinsufficiency for NF1. Studies in mouse models and NF1 families have implicated modifier genes unlinked to NF1 in the severity of the disease and in susceptibility to astrocytoma and peripheral nerve sheath tumors. To determine if differences in Nf1 expression may contribute to the strain-specific effects on tumor predisposition, we examined the levels of Nf1 gene expression in mouse strains with differences in tumor susceptibility using quantitative polymerase chain reaction. The data presented in this paper demonstrate that strain background has as much effect on Nf1 expression levels as mutation of one Nf1 allele, indicating that studies of haploinsufficiency must be carefully interpreted with respect to strain background. Because expression levels do not correlate entirely with the susceptibility or resistance to tumors observed in the strain, these data suggest that either variation in Nf1 levels is not responsible for the differences in astrocytoma and peripheral nerve sheath tumor susceptibility in Nf1-/+;Trp53-/+cis mice, or that certain mouse strains have evolved compensatory mechanisms for differences in Nf1 expression.

Linkage disequilibrium and founder effect analysis of the NF1 gene in French Canadians from the Quebec population

We genotyped 19 neurofibromatosis type 1 (NF1) families from French Canadians of the Quebec population with four intragenic microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Linkage analysis of the four microsatellite markers among the 19 NF1 families indicates that the four microsatellites are strongly linked with NF1 disease (LOD = 2.76–3.64). The four markers are associated ( P = 0–0.077) except marker pair IVS26-2.3/IVS27AC33.1 ( P = 0.18 or 0.17). However, perhaps due to the high mutation rate of the NF1 gene, no founder effect for NF1 was detected in the Quebec French Canadians.

Gross deletions of the neurofibromatosis type 1 (NF1) gene are predominantly of maternal origin and commonly associated with a learning disability, dysmorphic features and developmental delay.

Mutation screening in neurofibromatosis type 1 (NF1) families has long been hampered by the complexity of the NF1 gene. By using a novel multi-track screening strategy, 67 NF1 families (54 two-generation, 13 three-generation) with a de novo mutation in the germline of the first generation were studied with two extragenic and 11 intragenic markers. The pathological lesion was identified in 31 cases. Loss of heterozygosity (LOH) in the affected individual revealed a gross gene deletion in 15 of the two-generation families; in 12 (80%) of them, the deletion was maternally derived. Eleven patients with a gross deletion exhibited developmental delay, ten had dysmorphic features and six manifested a learning disability. No gross deletion was apparent in any of the 13 three-generation families, suggesting that such lesions are subject to more intense selection. In these families, the new mutation was of paternal origin in 11 kindreds and the underlying mutational event could be characterised in three of them.

Serum mitogenic activity on in vitro glial cells in Neurofibromatosis type 1

Glial mitogenic effect was investigated in sera from the following groups of subjects: group (1) 31 patients clinically and genetically affected by Neurofibromatosis type 1 (NF1) belonging to different families; group (2) 42 patients without family history of NF1 affected by sporadic neoplasms of the same histogenetic origin as the proliferative lesions that are present in NF1; group (3) 51 healthy volunteers without family history of NF1 nor of neoplastic disease; group (4) 54 clinically healthy relatives of the NF1 patients included in the first group. All NF1 patients and 3/54 healthy relatives had alterations of exons 31 or 32 of NF1 gene. Glial proliferation, measured by [ 3H]thymidine incorporation, was significantly increased by sera from all NF1 patients and from 23/54 of clinically healthy relatives, as compared to sera from healthy volunteers. This serum mitogenic activity strongly suggests the existence of soluble glial proliferating molecules in NF1 families. The molecular weight (3–30 kDa), the heat- and freeze-stability and the specificity for glial cells, suggest that the molecules responsible for this mitogenic effect are different from the growth factors previously described in NF1-associated tumor extracts and from lymphokines. Within each NF1 family, the maximal serum dilution stimulating glial proliferation was similar both in affected members and in their clinically healthy relatives. Since none of the clinically healthy relatives showing serum mitogenic activity was positive for the NF1 mutation analysis and, conversely, those having altered exons 31 or 32 of NF1 gene did not show any mitogenic activity, these results suggest that the phenotype expression of NF1 might depend not only on the NF1 mutations per se, but also on other genetic or epigenetic factors, such as serum glial proliferating molecules.

Identification of de novo deletions at the NF1 gene: no preferential paternal origin and phenotypic analysis of patients.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. To date, a relatively small number of NF1 mutations have been characterized, thus precluding genotype-phenotype correlations. By genotyping 75 NF1 families, we have detected six hemizygous patients (two of whom are members of the same family). The five presumed deletions were confirmed by two quantitative methods of analysis of NF1 copy number: Southern hybridization with cDNA probes and a single-strand conformation polymorphism analysis that discriminates between the NF1 gene and the pseudogene sequences. The five deletions remove most of the NF1 gene, at least 225 kb, from exon 9 to the 3' end of the coding sequence. The origin of de novo mutations in the NF1 gene has been reported to be mainly paternal but we have determined that four of the de novo deletions involved the maternal chromosome and one the paternal chromosome. The six patients with deletions exhibited precocious, multiple clinical features of the disease. The incidence of tumor complications, particularly plexiform neurofibromas and intracranial tumors, among this group of patients is higher than the observed incidence in our NF1 population, suggesting that NF1 haploinsufficiency may cause a more severe phenotype with regard to tumor development. In contrast to other reports that associated large deletions with mildly dysmorphic facies, mental retardation and a large number of cutaneous neurofibromas, only one out of our six patients presented this phenotype.

Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a prevalence of around 1 in 3500, affecting all ethnic groups. The clinical manifestations of the disease are variable, even among members of the same family, and affect a variety of tissues and cell types, including skin, iris, central and peripheral nervous systems and skeletal system. It has been reported that the majority of sporadic mutations in NF1 arise in paternally inherited alleles. We present here a collaborative study of the parental origin and type of mutation in individuals with de novo NF1, who account for up to a half of all cases of clinically diagnosed NF1. We have studied intragenic and extragenic markers in 470 NF1 families. In 32 of these families it was possible to assess the parental origin of a de novo NF1 mutation either by linkage analysis (in families with three generations) or by the detection of an intragenic deletion in a sporadic NF1 case. Eleven of these 32 families have three generations (the second and third generation being affected), with the mutation (not a large deletion) being of paternal origin in 82% of them (P < 0.05). In the other 21 families an intragenic deletion was detected, in 76% being in the maternal chromosome and in 24% in the paternal one (P < 0.05). Our results suggest that in NF1 the majority of deletions occur in oogenesis, while other types of mutations should account for the paternally derived NF1 mutations.

Linkage disequilibrium between four intragenic polymorphic microsatellites of the NF1 gene and its implications for genetic counselling.

Four intragenic polymorphic microsatellite markers, AAAT Alu repeat, IVS27AC28.4, ACI27.2, and IVS38GT53.0, located along a 65 kb DNA region of the NF1 gene, were used to genotype 64 Spanish families with neurofibromatosis type 1 (NF1). Linkage disequilirium between each pair of markers was evaluated. Three of these markers, AAAT Alu repeat, ACI27.2, and IVS38GT53.0, exhibit linkage disequilibrium between each other. Analysis of extended haplotypes provides further evidence of the disequilibrium within this region since only 11 haplotypes account for 52% of the total chromosomes. Because of linkage disequilibrium, the informativeness of marker combinations for genotyping of NF1 families is diminished. There was no difference in the overall distribution of alleles between affected and normal chromosomes. An at risk haplotype was not found, as expected for a disease with at least 50% of cases being sporadic.

Neurofibromatosis/Noonan phenotype: a variable feature of type 1 neurofibromatosis.

Since January 1989 we have ascertained patients with neurofibromatosis type 1 (NF1) as part of our genetic register in the North West of England. This register has now identified 453 affected cases from 235 families. The first 94 individuals were specifically examined for features of the Noonan phenotype. This was present in 12/94 sequentially identified individuals with NF1 including six individuals from three families. However, three cases occurred in a further family, where Noonan syndrome appeared to segregate separately from NF1. We have provided evidence for the chance association of Noonan syndrome and NF1 and that the Noonan phenotype occurs as a feature in some NF1 families. However, there is now little evidence of a separate NF1/Noonan syndrome entity or of NF1 features occurring in classical Noonan syndrome.

Two single base polymorphisms in introns 41 and 16 of the NF1 gene.

We have characterized two intragenic polymorphisms in the neurofibromatosis type 1 (NF1) gene by direct sequencing of PCR products. The variants for these polymorphisms were initially detected on Hydrolink gels. One of the polymorphisms involves a G to A transition in intron 41 at the 28th base upstream of exon 42 with an observed 'G'/'A' heterozygosity of 0.42. The other polymorphism is a T to C transition in intron 16 at the 16th base upstream of exon 17 with an observed 'T'/'C' heterozygosity of 0.09. In combination with other documented polymorphisms in the NF1 gene, these variants should assist in genetic analysis of NF1 families.

Prenatal diagnosis of neurofibromatosis type 1: From flanking rflps to intragenic microsatellite markers

Even though the neurofibromatosis type 1 (NF1) gene was cloned more than 3 years ago, the process of identifying mutations has not been fruitful, and genetic counselling is mainly based on the use of linked markers. Since 1990, we have analysed 130 NF1 families and have performed six prenatal diagnoses. In each case, genetic counselling has relied on linked markers and informativity was achieved in all of them. The use of intragenic microsatellite polymorphisms (IVS27AAAT2.1, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0) has increased the informativeness in our series of NF1 families to an average of 90 per cent, providing accurate diagnosis and confirmation of the disease status.