Mutations In NF1 Gene Research Articles

655 ISOLATED PHAECHROMOCYTOMA

Introduction: Phaechromocytoma is a neuroendocrine tumor associated with a variety of genetic disorders wich include(VHL),multiple endocrine neoplasia type2,Neurofibromatosis type 1 and hereditary pargangliomas.These hereditary syndrome are respectively caused by respectively mutation in VHL tumor suppressor gene,RET protooncogene,NF1 gene and gene coding succinate deshydrogenase SDHB,SDHC and SDHD.It is currently estimated that 20% of phaechromacytoma are associated with germline mutation.VHL predispose affected individual to develop retinal and central nervous hemangioblastoma, renal celle carcinoma and phaechromocytoma. We report a novel VHL germ-line mutation in a family with occurrence of isolated phaechromocytoma. Case: The 38 years old, caucasian male was admitted in hospital for major weakness, sweating ; body loss,elevated pressure, moderate renal failure ;Hypokalaemia. Plasma renine aldosterone activity is high, plasma fractionnal free metanephrine is very high Proteinuria :3 gr/24 h, microscopic haematuria. urinary normetadrenaline was very high 15916ng/24 h (normal < 200). Radiologic test: Brain, spinal, abdominal and thoraco abdominal CT scan, showed bilateral surrenale mass, left measured 4.5*3.5*4 cm, right 4.5*4*4.4 cm .MIBG scintigraphy : cervical para ganglioma. Patient have had bilateral adrenalectomy. Genetic analysis: VHL germline mutation was identified in the patient and family members.The c.640T > A mutation detected in our reported family has not been previously described. Discussion: This patient had presented phaechromocytoma in association and without other manifestation of VHL disease. The association of this mutation (c.640 > A) with phaechromocytoma only, variant of VHL disease, might be considered. To confirm this suggestion it seems reasonnable to subject these patient to lifelong follow up.

A novel NF1 mutation in a Chinese patient with giant café-au-lait macule in neurofibromatosis type 1 associated with a malignant peripheral nerve sheath tumor and bone abnormality

Neurofibromatosis type 1 (NF1; OMIM#162200) is a common neurocutaneous disorder that is characterized by multiple café-au-lait, skinfold freckling, Lisch nodules, and neurofibromas. Mutations in the NF1 gene, which encodes the neurofibromin protein, have been identified as the pathogenic gene of NF1. In this study, we present a clinical and molecular study of a Chinese patient with giant café-au-lait in NF1. The patient showed >6 café-au-lait spots on the body, axillary freckling, and multiple subcutaneous neurofibromas. He also had a malignant peripheral nerve sheath tumor and bone abnormalities. The germline mutational analysis of the NF1 gene revealed a novel missense mutation in exon 13. It is a novel heterozygous nucleotide G>A transition at position 2241 of the NF1 gene. We found no mutation in malignant peripheral nerve sheath tumor DNA from this patient. This expands the database for NF1 gene mutations in NF1. Its absence in the normal chromosomes suggests that it is responsible for the NF1 phenotype. To our knowledge, this is the first case of giant café-au-lait macule in NF1 associated with a malignant peripheral nerve sheath tumor and bone abnormality.

Two pathogenic NF1 gene mutations identified in DNA from a child with mild phenotype

Two pathogenic NF1 gene mutations identified in DNA from a child with mild phenotype

Patient-Derived Induced Pluripotent Stem Cells Recapitulate Hematopoietic Abnormalities of Juvenile Myelomonocytic Leukemia

Abstract 637Juvenile Myelomonocytic Leukemia (JMML) is an aggressive childhood myeloproliferative disorder caused by NF1, NRAS, KRAS, PTPN11 or CBL gene mutations that induce Ras pathway activation with associated hypersensitivity to cytokine stimulation in myeloid progenitor cells. Understanding the pathophysiology of JMML and developing new treatments is constrained by limited access to relevant patient material. To address this problem, we generated induced pluripotent stem cells (iPSCs) from normal neonatal umbilical cord blood and two JMML patients with different mutations: 1) a somatic heterozygous E76K substitution in PTPN11 and 2) a CBL Y371H substitution arising from a germline mutation. We created iPSCs from the patient's heterozygous CBL Y371H newborn cord blood cells prior to the diagnosis of JMML and then from peripheral blood at age 7 months, when JMML ensued with outgrowth of CBL Y371H homozygous malignant cells. We reprogrammed control and malignant cells using the STEMCCA lentiviral vector system that expresses OCT4, KLF4, MYC and SOX2 in a doxycyline-inducible fashion. Resultant iPSCs exhibited hallmark features after more than twenty passages, including characteristic morphology, expression of endogenous pluripotency markers, silencing of viral reprogramming genes in the absence of doxycycline, normal karyotype and formation of endoderm-, ectoderm- and mesoderm-derived tissues in teratoma assays. Relevant PTPN11 and CBL genotypes of these clones were confirmed by DNA sequencing.Selected iPSC clones were differentiated into blood by inducing the formation of embryoid bodies in serum free medium with defined cytokines. By day 8–9, hematopoietic progenitors (CD43+, CD235+, CD41+) with erythroid, megakaryocytic and myeloid potential developed from both control (n = 2 lines) and JMML iPSCs (n= 2 lines from each patient). In methylcellulose colony assays, JMML progenitors exhibited relative hypersensitivity to GM-CSF, as reflected by increased numbers and larger size of myeloid colonies at limiting GM-CSF concentrations. In liquid cultures containing SCF, TPO, EPO, IL-3, IL-11 and IGF-1, with or without GM-CSF, the JMML progenitors produced increased proportions of CD33+CD14+ myelomonocytic cells compared to controls. Moreover, in GM-CSF dose-response assays, single cell phospho-flow cytometry analysis showed sustained STAT5 activation in JMML myeloid cells vs. controls. Thus, key pathological features of JMML, including propensity to myelomonocytic cell expansion and GM-CSF hypersensitivity, are recapitulated by in vitro differentiation of JMML iPSCs. Our findings illustrate the utility of iPSCs for modeling human blood disorders and more specifically, provide renewable sources of biologically relevant, patient-derived cells in which to explore the pathophysiology and therapy of JMML. Disclosures:No relevant conflicts of interest to declare.

Successful application of the strategy of blastocyst biopsy, vitrification, whole genome amplification, and thawed embryo transfer for preimplantation genetic diagnosis of neurofibromatosis type 1

Objective Preimplantation genetic diagnosis (PGD) offers an alternative for women to carry an unaffected fetus risk of hereditary diseases. Trophectoderm biopsy may provide more cells for accurate diagnosis. However, the time allowed for transportation of the specimens to the laboratory and performance of molecular diagnosis is limited. We designed a PGD program of trophectoderm biopsy, vitrification of blastocysts, whole genome amplification (WGA), double confirmatory genotypings, and thawed embryo transfer. Case Report We conducted this strategy for a woman of familial neurofibromatosis type I (NF-1). She had a genotype of heterozygous c.6709C>T mutation of NF1 gene. Trophectoderm biopsies were performed on 13 blastocysts. Then, individual blastocyst was vitrified. WGA was performed for the samples, followed by genotypings with both real-time polymerase chain reaction and sequencing. Eight embryos were diagnosed as unaffected, four were affected, and one was inconclusive because of allele drop-out. In the next cycle, two unaffected blastocysts were thawed and transferred, that resulted in a singleton pregnancy. The pregnancy was confirmed as unaffected by means of chorionic villi sampling. Conclusion We first demonstrate successful application of blastocyst biopsy, vitrification, WGA, and thawed embryo transfer for PGD of a monogenic disease. Vitrification of blastocysts after biopsy permits sufficient time for shipment of samples and operation of molecular diagnosis.

Malignancies Associated With Germline And Somatic NF1 Gene Mutations

Malignancies Associated With Germline And Somatic NF1 Gene Mutations

Mutation analysis of NF2 gene and clinical investigation in a Chinese family with neurofibromatosis type II

To report a heterozygous RNA-splicing mutation (IVS3+ 3A to C) of NF2 gene in a Chinese family with autosomal dominant neurofibromatosis type II and investigate the relationship between the genotype and phenotype. The proband with bilateral vestibular schwannomas underwent gamma knife radiosurgery two years earlier. DNA of blood samples from all affected individuals, suspected individuals and unaffected relatives of the family was extracted and amplified to detect the polymorphisms at loci D22S1150 and D22S268 that are linked with the NF2 gene. Two-point LOD score was calculated. The promoter region, 17 exons and exon/intron boundaries of NF2 gene were amplified and sequenced for the proband. The exon 3/intron 3 boundaries of NF2 gene was amplified and sequenced for the other 3 patients, 1 suspected individual, 9 unaffected members of the family and 150 unrelated controls. The result of two-point linkage analysis suggested that NF2 gene was a candidate gene (Zmax= 2.109, θ = 0.00, locus D22S1150). DNA sequencing revealed a heterozygous splicing mutation in intron 3 (IVS3+ 3A to C) for the proband. Identical mutation was also observed in the other 3 patients and 1 suspected individual. No mutation was found in the 9 normal family members and 150 unrelated controls, which was consistent with the clinical diagnosis. This is the first report of familial neurofibromatosis type II with a splicing mutation of IVS3+ 3A to C of the NF2 gene. The mutation might be responsible for the neurofibromatosis type II in the family.

Neurofibromatosis type 2: Molecular and clinical analyses in Argentine sporadic and familial cases

Neurofibromatosis 2 is a familial syndrome characterized by the development of schwannomas, meningiomas and ependymomas. Most of them are benign however, their location in the nervous system has harmful effects on important cranial and spinal structures. These tumors are developed as the outcome of NF2 gene (22q12) inactivation. The NF2 protein, merlin or schwannomin belongs to the Ezrin, Radixin, Moesin (ERM) family involved in the cytoskeletal network and has a tumor suppressor function. Inactivating mutations occur as “de novo” (more frequently) or as inherited, and most of them are frameshift or nonsense. Our aim is to study NF2 gene alterations in Argentine patients and relate them to clinical features. 10 families and 29 single patients were analyzed for: 1) at-risk haplotype by STR-segregation analysis and 2) NF2 gene mutations by SSCP/heteroduplex/sequencing. The at-risk haplotype was uncovered in 8 families and mutations were identified in 5 patients. The molecular data are in full agreement with the clinical features supporting previous reports. The obtained results were important for the detection of mutation-carrying relatives and exclusion of other individuals from risk.

Analysis of NF1 somatic mutations in cutaneous neurofibromas from patients with high tumor burden

Neurofibromatosis type 1, (NF1) is a complex, autosomal dominant disorder characterized by benign and malignant tumors which result from NF1 gene mutations. The molecular mechanisms that underlie NF1 tumorigenesis are still poorly understood although inactivation of other modifying loci in conjunction with NF1 mutations is postulated to be involved. These modifying loci may include deficiencies in mismatch repair genes and elements involved in cell cycle regulation (TP53, RB1, and CDKN2A). We have analyzed the somatic mutations in 89 cutaneous neurofibromas derived from three unrelated NF1 patients with high tumor burden, by loss of heterozygosity (LOH) analysis of the NF1, TP53, RB1, and CDKN2A genes, by assessing microsatellite instability (MSI), by direct sequencing of the NF1, TP53, and several mismatch repair (MMR) genes and by multiplex ligation-dependent probe amplification of the NF1 and TP53 genes. The aim was both to assess the possible clonality of these tumors and also to assess the involvement of other potential genetic loci in the development of these neurofibromas. Somatic NF1 mutations were identified in 57 (64%) of neurofibroma samples. Each mutation was distinct demonstrating the independent origin of each tumor. While somatic LOH of the TP53 gene was identified in four tumors, no specific deletions or sequence variations were identified. LOH of markers flanking the RB1 gene was also found in one tumor but no CDKN2A mutations were detected. Although evidence of MSI was seen in 21 tumors, no MMR gene alterations were identified. The identification of LOH involving TP53 and RB1 loci is a novel finding in benign cutaneous neurofibromas possibly demonstrating an alternative underlying molecular mechanism associated with the development of these benign tumors from this cohort of patients.

An infrequent plexiform variant of schwannoma of the glans penis: a rare finding

An infrequent plexiform variant of schwannoma of the glans penis: a rare finding

Somatic alterations of the NF1 gene in an NF1 individual with multiple benign tumours (internal and external) and malignant tumour types

Neurofibromatosis type 1 is a common familial cancer syndrome, affecting about 1 in every 4,000 individuals worldwide. We have carried out NF1 gene mutation analysis on DNA isolated from 25 tumours (dermal and plexiform neurofibromas, malignant peripheral nerve sheath tumour, MPNST), obtained at post-mortem from an NF1 patient. Macro and micro sequence alterations of the NF1 gene were studied by dHPLC, microsatellite, RFLP markers and multiplex ligation probe amplification (MLPA). The underlying germline mutation involves a deletion of exons 2 and 3. Of the 25 tumours studied from this patient, characterised somatic mutations were identified in 9 tumours, these were six small deletions (748del T, 2534-2557 del 24bp, 2843delA, 3047-3048 del GT, 4743del G, 7720-7721 delAA), an insertion 649 ins 73 bp, a non-sense mutation R1513X and a single splice site mutation, IVS4C-1 G>A, eight of these represent novel sequence changes in the gene. Evidence for loss of heterozygosity (LOH) was identified in DNA from 7 of the tumours. Each of the tumours analysed contained a different somatic NF1 mutation, indicating that each tumour is the result of an independent somatic event. The somatic mutation detection rate in this study is 64% (16/25), is one of the highest rates in genomic DNA reported so far in a single NF1 patient. Only 68 characterised NF1 somatic mutations have so far been reported and so our data will contribute to NF1 somatic mutational spectrum of the NF1 gene and will be important for understanding the molecular basis of NF1 tumorigenesis.

Germline Missense Mutations Affecting KRAS Isoform B Are Associated with a Severe Noonan Syndrome Phenotype

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring-binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor.

Molecular genetics alterations and tumor behavior of sporadic vestibular schwannoma from the People’s Republic of China

To analyze the molecular genetic alteration of sporadic vestibular schwannomas from the People's Republic of China and to correlate these alterations with the tumor behaviors. Four highly polymorphic microsatellite DNA markers were used to observe the frequency of loss of heterozygosity (LOH) in chromosome 22. The NF2 gene mutations were detected by Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. The schwannomin/merlin (S/M) expression was examined using anti-NF2 (A-19) IgG under immunohistochemistry and western blot. The proliferative index (LI) of vestibular schwannoma was evaluated by proliferative cell nuclear antigen investigation. Sixteen vestibular schwannomas (44.4%) showed allele loss. We found 22 mutations in 36 schwannomas. The LI and the growth rate of schwannomas with LOH or mutation were significantly higher than those without LOH or mutation. All of these vestibular schwannomas showed no immunoreaction to anti-NF2(A-19) IgG by immunohistochemistry. By immunoblotting technique, reduced expression of S/M was found in 31 cases (86%). The growth index of schwannomas with severely reduced expression of S/M was significantly higher than those with moderately reduced or normal expression. The molecular genetic changes in sporadic vestibular schwannomas from Chinese patients were similar to the previous reports. We demonstrate the relationship between tumor behaviors and genetic alteration (including LOH and mutation of NF2 gene). We propose that inactivation of S/M, may be an important step in tumorigenesis of sporadic vestibular schwannoma.

Subfrontal schwannoma

Subfrontal schwannoma is a rare disease, which can be mis-diagnosed as an olfactory meningioma or a neuroblastoma, because of similar clinical symptoms and signs and neuroradiological features. Especially for young subjects, olfactory neuroblastoma should be carefully differentiated, since the management strategies for those lesions are significantly different. The craniofacial approach is often needed for the resection of a neuroblastoma. We report a case of 14-year old boy in which olfactory neuroblastoma was suspected prior to surgery, but turned out to be a schwannoma histologically. Molecular genetic examination revealed neither NF2 gene mutation nor loss of heterozygosity of chromosome 22q, unlike common schwannomas.

Genetic and epigenetic alteration of the NF2 gene in sporadic meningiomas

The role of the NF2 gene in the development of meningiomas has recently been documented; inactivating mutations plus allelic loss at 22q, the site of this gene (at 22q12), have been identified in both sporadic and neurofibromatosis type 2-associated tumors. Although epigenetic inactivation through aberrant CpG island methylation of the NF2 5' flanking region has been documented in schwannoma (another NF2-associated neoplasm), data on participation of this epigenetic modification in meningiomas are not yet widely available. Using methylation-specific PCR (MSP) plus sequencing, we assessed the presence of aberrant promoter NF2 methylation in a series of 88 meningiomas (61 grade I, 24 grade II, and 3 grade III), in which the allelic constitution at 22q and the NF2 mutational status also were determined by RFLP/microsatellite and PCR-SSCP analyses. Chromosome 22 allelic loss, NF2 gene mutation, and aberrant NF2 promoter methylation were detected in 49%, 24%, and 26% of cases, respectively. Aberrant NF2 methylation with loss of heterozygosity (LOH) at 22q was found in five cases, and aberrant methylation with NF2 mutation in another; LOH 22q and the mutation were found in 16 samples. The aberrant methylation of the NF2 gene also was the sole alteration in 15 samples, most of which were from grade I tumors. These results indicate that aberrant NF2 hypermethylation may participate in the development of a significant proportion of sporadic meningiomas, primarily those of grade I.

Genetic Dissection of Cooperating Mutations in BXH-2 Acute Myeloid Leukemia with and without Nf1 Gene Mutation.

Loss of the NF1 (Neurofibromatosis Type 1) gene, a tumor suppressor, can cause myeloid diseases juvenile myelomonocytic leukemia (JMML), monosomy 7 syndrome (Mo7), and acute myeloid leukemia (AML). However, using knockout mice, it has been shown that loss of Nf1 expression in hematopoietic cells, by itself, does not lead to aggressive leukemia resulting instead in a relatively indolent myeloproliferative disease. Murine Leukemia Virus (MuLV) insertional mutagenesis in BXH-2 mice provides a model to dissect genetic alterations in AML. We have profiled proviral insertions in BXH-2 AML which do or do not have corresponding loss-of-function of Nf1. 197 PIS (68 from 25 Nf1-wild type AML and 129 from 55 Nf1-defective AML) were isolated. Nf1-defective AML were obtained from BXH-2 AMLs with proviral insertions into the endogenous Nf1 gene and AML that developed in leukemia-prone, heterozygous Nf1+/− BXH-2 mice. These latter AMLs develop faster than wild-type BXH-2 AMLs and show Nf1 gene LOH or proviral insertion into the wild-type Nf1 allele. These analyses led to 37 common proviral insertion sites (CIS), 13 of which have not been reported previously. Several of the CIS (including Lmo2, Cmyb, Meis1, Bcl11a, Spred2, Def8, Edg3, Hoxa9, and a novel Krab domain-zinc finger gene) were found repeatedly among the Nf1-defective group of AML. Expression of most could be detected in human JMML and CMML by RT-PCR, including BCL11A. Importantly, among the CIS we detected, PIS targeting Bcl11a were significantly enriched (p < 0.05) in Nf1-defective leukemia. Retroviral expression vectors for Bcl11a have been constructed and transduced into an immortalized Nf1-/- null myeloblast cell line. Growth assays show that the cumulative cell number of FACS-sorted Bcl11a-Nf1-/- cells increase by ~2.5 fold that of controls. BXH-2 provides a powerful genetic system to dissect Nf1-cooperating genetic events in tumorigenesis. Mutations at several novel common integration sites could be involved in development or progression of leukemia with NF1 gene inactivation. This work was supported by the National Cancer Institute (U01-CA84221-05) and the American Cancer Society (RPG LIB-106632) to DAL and by National Cancer Institute (R01 CA92095) and U.S. Dept. of Defense (DAMD17-97-1-7339) to MRW.

Familial Segmental Neurofibromatosis

Segmental neurofibromatosis is considered to be the result of postzygotic NF1 gene mutations. We present a family in which the proband has generalized neurofibromatosis 1, whereas members of previous generations manifest segmental skin lesions. All, including the clinically asymptomatic grandmother, carry the same haplotype. This is the only case in the literature in which a parent with segmental skin findings has a child with full-blown neurofibromatosis 1 disease. The genetic mechanisms underlying this association are discussed. This family can be further investigated by examination of tissue samples from affected and unaffected sites for mutations.

Neurofibromatosis type 1 (NF1): Identification of eight unreported mutations in NF1 gene in Italian patients [corrected

In the present study the entire NF1 coding region was analyzed for mutations in 132 unrelated Italian NF1 patients. Using PTT, SSCP, and DNA sequencing, we found 8 novel mutations. Clinical diagnosis of NF1 was established according to the NIH consensus criteria. We detected 59 truncated fragments, and 46 of them were characterized by SSCP and direct sequencing. Eight mutations represent novel changes that contribute to the germline mutational spectrum of the NF1 gene. In two patients, premature termination was due to substitutions at nucleotide c.3982C>T (Q1298X) and c.7411C>T (Q2471X), respectively. Two other mutations were caused by the deletions (1756delA, 4699delA), and two by the insertions (c.5266_5267insT, c.7464_7465insTCCA) of a small number of nucleotides. Lastly, we found 2 splice-site mutations (c.2252-2A>C, c.2251+1G>A).

Hemizygosity of Nf2 is associated with increased susceptibility to asbestos-induced peritoneal tumours.

Biallelic NF2 gene inactivation is frequently found in human malignant mesothelioma. In order to assess whether NF2 hemizygosity may enhance susceptibility to asbestos fibres, we investigated the Nf2 status in mesothelioma developed in mice presenting a heterozygous mutation of the Nf2 gene (Nf2(KO3/+)), after intraperitoneal inoculation of crocidolite fibres. Asbestos-exposed Nf2(KO3/+) mice developed tumoural ascites and mesothelioma at a higher frequency than their wild-type (WT) counterparts (P&<0.05). Six out of seven mesothelioma cell lines established from neoplastic ascitic fluids of Nf2(KO3/+) mice exhibited loss of the WT Nf2 allele and no neurofibromatosis type 2 protein expression was found in these cells. The results show the importance of the NF2 gene in mesothelial oncogenesis, the potential association of asbestos exposure and tumour suppressor gene inactivation, and suggest that NF2 gene mutation may be a susceptibility factor to asbestos.

Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis.

BackgroundNF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes.MethodsThe expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization.ResultsThe expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins.ConclusionsThese results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes.