Normal Neurofibromatosis Type 1 Allele Research Articles

Monozygotic Twins With Neurofibromatosis Type 1 (NF1) Display Differences in Methylation ofNF1Gene Promoter Elements, 5' Untranslated region, Exon and Intron 1

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder caused by heterozygotic inactivation of the NF1 tumor suppressor gene at 17q11.2. The associated phenotypes are highly variable, and modifying genes have been proposed to explain at least in part the intriguing expressivity. Given that haploinsufficiency of the NF1 gene product neurofibromin is responsible for some of the clinical manifestations, variations in expression of the wildtype NF1 allele might modify the phenotype. We therefore investigated epigenetic molecular modifications that could result in variable expression of the normal NF1 allele. To exclude confounding by DNA sequence variations, we analyzed monozygotic twin pairs with NF1 who presented with several discordant features. We fine-mapped the methylation pattern of a nearly 1 kb NF1 promoter region in lymphocytes of 8 twin pairs. All twin pairs showed significant intra-pair differences in methylation, especially of specific promoter subregions such as 5'UTR, exon 1 and intron 1 (+7 to +622), transcription factor binding sites and promoter elements like NF1HCS. Furthermore, we detected significant intra-pair differences in cytosine methylation for the region from -249 to -234 with regard to discordance for optic glioma with a higher grade of methylation in glioma cases. In conclusion, our findings of epigenetic differences of the NF1 promoter in leukocytes within mono zygotic twin pairs may serve as a proof of principle for other tissues. The results point towards a role of methylation patterns of the normal NF1 allele for expression differences and for modification of the NF1 phenotype.

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.

An Absence of Cutaneous Neurofibromas Associated with a 3-bp Inframe Deletion in Exon 17 of the NF1 Gene (c.2970-2972 delAAT): Evidence of a Clinically Significant NF1 Genotype-Phenotype Correlation

Neurofibromatosis type 1 (NF1) is characterized by cafe-au-lait spots, skinfold freckling, and cutaneous neurofibromas. No obvious relationships between small mutations (<20 bp) of the NF1 gene and a specific phenotype have previously been demonstrated, which suggests that interaction with either unlinked modifying genes and/or the normal NF1 allele may be involved in the development of the particular clinical features associated with NF1. We identified 21 unrelated probands with NF1 (14 familial and 7 sporadic cases) who were all found to have the same c.2970-2972 delAAT (p.990delM) mutation but no cutaneous neurofibromas or clinically obvious plexiform neurofibromas. Molecular analysis identified the same 3-bp inframe deletion (c.2970-2972 delAAT) in exon 17 of the NF1 gene in all affected subjects. The Delta AAT mutation is predicted to result in the loss of one of two adjacent methionines (codon 991 or 992) ( Delta Met991), in conjunction with silent ACA-->ACG change of codon 990. These two methionine residues are located in a highly conserved region of neurofibromin and are expected, therefore, to have a functional role in the protein. Our data represent results from the first study to correlate a specific small mutation of the NF1 gene to the expression of a particular clinical phenotype. The biological mechanism that relates this specific mutation to the suppression of cutaneous neurofibroma development is unknown.

Genetic Disruption of Rac1 but Not Rac2 Is Sufficient To Ameliorate the Development of Juvenile Myeloproliferative Myeloid Leukemia in Nf1−/− Mice.

Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21ras proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile myelomonocytic leukemia (JMML) associated with loss of the normal NF1 allele. Genetically engineered mice containing nullizygous loss of Nf1 in the hematopoietic system develop a leukemia with complete penetrance that is similar to JMML, including hepatosplenomegaly, elevated peripheral blood cell counts, and elevated numbers of myeloid progenitors that are hypersensitive to multiple cytokines, particularly GM-CSF. Though we and others have found that p21ras is hyperactivated in Nf1−/− myeloid progenitors, inhibiting Ras specifically as a therapeutic target has been challenging. Therefore, identification of alterations in distinct p21ras effector pathways that control leukemia progression in Nf1-deficient cells is critical for understanding disease pathogenesis and identifying therapeutic targets. Here we intercrossed MxCre; Nf1flox/flox mice with mice that are deficient in the small Rho GTPases Rac1 or Rac2 to generate syngeneic progeny that were MxCre; Nf1flox/flox, MxCre; Nf1flox/flox;Rac1flox flox or MxCre; Nf1flox/flox; Rac2 −/−. Consistent with previous studies (Le, Blood 2004) MxCre; Nf1flox/flox mice develop a progressive myeloproliferative disease with 100% penetrance 6 months following interferon inducible induction of the MxCre transgene to disrupt the Nf1flox alleles. Eighty percent of MxCre; Nf1flox/flox die by 9 months after inactivation. Genetic disruption of Rac2 was not sufficient to diminish the onset or severity of the characteristic myeloproliferative disease of MxCre; Nf1flox/flox mice. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice followed for 11 months all survived and had normal bone marrow cellularity, spleen weight and splenic architecture. MxCre; Nf1flox/flox mice have elevated numbers of both HPP-CFC and LPP-CFC in the bone marrow and spleen. In contrast, MxCre; Nf1flox/flox; Rac1flox/flox mice had myeloid progenitor numbers that were comparable to wildtype, age-matched controls. Further, though myeloid progenitors from MxCre; Nf1flox/flox mice are hypersensitive to GM-CSF, myeloid progenitors from MxCre; Nf1flox/flox; Rac1flox/flox have a sensitivity to GM-CSF comparable to wildtype controls. The correction in the myeloproliferative phenotype isolated from MxCre; Nf1flox/flox; Rac1flox/flox mice was associated with the reduction in Rac-GTP and prolonged Erk phosphorylation, a MAPK effector that is characteristically elevated in Nf1−/− myeloid progenitors. Collectively, these genetic data identify Ras-Rac1 signaling pathway as a key axis in the genesis of juvenile myelomonocytic leukemia and provide evidence that Rac1 is a therapeutic molecular target for this myeloproliferative disease that currently has no effective therapies.

Evidence that juvenile myelomonocytic leukemia can arise from a pluripotential stem cell

AbstractChildren with neurofibromatosis type 1 (NF1) carry germline mutations in one allele of the NF1 gene and are predisposed to myeloid malignancies, particularly juvenile myelomonocytic leukemia (JMML). Disruption of the remaining NF1 allele can be found in malignant cells. Flow cytometric cell sorting techniques to isolate the malignant cell populations and molecular genetic methods to assay for somatic loss of the normal NF1 allele were used to study an unusual child with NF1 and JMML who subsequently had T-cell lymphoma. The data show that malignant JMML and lymphoma cells share a common loss of genetic material involving the normal NF1gene and approximately 50 Mb of flanking sequence, suggesting that the abnormal T-lymphoid and myeloid populations were derived from a common precursor cell. These data support the hypothesis that JMML can arise in a pluripotent hematopoietic cell.

Evidence that juvenile myelomonocytic leukemia can arise from a pluripotential stem cell

Children with neurofibromatosis type 1 (NF1) carry germline mutations in one allele of the NF1 gene and are predisposed to myeloid malignancies, particularly juvenile myelomonocytic leukemia (JMML). Disruption of the remaining NF1 allele can be found in malignant cells. Flow cytometric cell sorting techniques to isolate the malignant cell populations and molecular genetic methods to assay for somatic loss of the normal NF1 allele were used to study an unusual child with NF1 and JMML who subsequently had T-cell lymphoma. The data show that malignant JMML and lymphoma cells share a common loss of genetic material involving the normal NF1 gene and approximately 50 Mb of flanking sequence, suggesting that the abnormal T-lymphoid and myeloid populations were derived from a common precursor cell. These data support the hypothesis that JMML can arise in a pluripotent hematopoietic cell.

Genetic and cellular defects contributing to benign tumor formation in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common inherited cancer predisposition syndrome. The NF1 gene product, neurofibromin, is hypothesized to function as a tumor suppressor and nearly all NF1 patients develop benign peripheral nerve tumors. These neurofibromas presumably arise from NF1 inactivation in S100(+)Schwann cells, but there is no formal proof for this mechanism. We demonstrate that fibro-blasts isolated from neurofibromas carried at least one normal NF1 allele and expressed both NF1 mRNA and protein, whereas the S100(+)cells typically lacked the NF1 transcript. Our findings further indicate that additional molecular events aside from NF1 inactivation in Schwann cells and/or other neural crest derivatives contribute to neurofibroma formation.

Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo.

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.

Hyperactive Ras as a therapeutic target in neurofibromatosis type 1

The NF1 gene encodes neurofibromin, a GTPase-activating protein (GAP) for members of the p21(ras) (Ras) family, which negatively regulates Ras output by accelerating the conversion of active Ras. GTP to inactive Ras.GDP. Analysis of tumors from patients with neurofibromatosis type 1 (NF1) has shown biochemical evidence of hyperactive Ras as well as frequent loss of the normal NF1 allele, consistent with its role as a tumor suppressor gene. Taken together, these data suggest that novel therapeutics directed against components of the Ras signaling cascade might provide effective treatments for certain pathological complications of NF1. Here we summarize data that support a role for hyperactive Ras in NF1 disease, including Ras processing, activation, and down-regulation. We review targets for rational drug design, provide preliminary results, and discuss implications for future studies. Am. J. Med. Genet. (Semin. Med. Genet.) 89:14-22, 1999.

Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders.

The risk of malignant myeloid disorders in young children with neurofibromatosis type 1 is 200 to 500 times the normal risk. The gene for neurofibromatosis type 1 (NF1) encodes neurofibromin, a protein that negatively regulates signals transduced by Ras proteins. Genetic and biochemical data support the hypothesis that NF1 functions as a tumor-suppressor gene in immature myeloid cells, but inactivation of both NF1 alleles has not been demonstrated in leukemic cells from patients with neurofibromatosis type 1. Using an in vitro transcription and translation system, we screened bone marrow samples from 18 children with neurofibromatosis type 1 and myeloid disorders for NF1 mutations that cause a truncated protein. Mutations were confirmed by direct sequencing of genomic DNA from the patients, and from their affected parents, in cases of familial neurofibromatosis type 1. Specimens from 9 of the 18 children contained abnormal peptide fragments, and truncating mutations of the NF1 gene were found in specimens from 8 of these children. The normal NF1 allele was absent in bone marrow samples from five of the eight children. We detected the same mutation in DNA from the affected parent of each child with familial neurofibromatosis type 1. Both alleles of the NF1 gene are inactivated in leukemic cells in some patients with neurofibromatosis type 1. NF1 appears to function as a tumor-suppressor gene in immature myeloid cells.

Monosomy 7 myelodysplastic syndrome and other second malignant neoplasms in children with neurofibromatosis type 1

Children with neurofibromatosis type 1 (NF1) are at increased risk of developing benign and malignant solid tumors as well as hematologic malignancies, including de novo juvenile chronic myelogenous leukemia, monosomy 7 syndrome, and acute myelogenous leukemia. The normal NF1 allele is frequently deleted in the bone marrow cells from NF1 patients with hematologic malignancies, suggesting a pathogenic role in primary leukemogenesis. The authors report monosomy 7 myelodysplastic syndrome (MDS) as a second malignant neoplasm (SMN) in five children with sporadic NF1, the results of molecular analysis for NF1 and ras alterations in their bone marrow, and summarize their experience with SMNs in pediatric patients with NF1. Monosomy 7 MDS was diagnosed as an SMN in five children with NF1 by morphologic and cytogenetic studies of bone marrow specimens. DNA extracted from these malignant bone marrows was analyzed for allelic loss at the NF1 locus and for the presence of ras mutations. The Children's Hospital of Philadelphia (CHOP) Tumor Registry was also reviewed to estimate the incidence of SMNs in pediatric NF1 patients. Bone marrow specimens from four patients retained constitutional heterozygosity at the NF1 locus; one specimen was uninformative. There was no evidence of activating ras mutations. The risk of an SMN was approximately 11% among the 64 NF1 registrants with primary malignancies in the CHOP registry, but was 75% (6 of 8) among patients treated for a pediatric embryonal cancer. Children with NF1 are susceptible to the development of malignant myeloid disorders both as a primary event and as an SMN. Additional molecular genetic analysis is necessary to determine if the NF1 gene is inactivated by somatic mutation in these secondary leukemias.

Monosomy 7 myelodysplastic syndrome and other second malignant neoplasms in children with neurofibromatosis type 1

BACKGROUND Children with neurofibromatosis type 1 (NF1) are at increased risk of developing benign and malignant solid tumors as well as hematologic malignancies, including de novo juvenile chronic myelogenous leukemia, monosomy 7 syndrome, and acute myelogenous leukemia. The normal NF1 allele is frequently deleted in the bone marrow cells from NF1 patients with hematologic malignancies, suggesting a pathogenic role in primary leukemogenesis. The authors report monosomy 7 myelodysplastic syndrome (MDS) as a second malignant neoplasm (SMN) in five children with sporadic NF1, the results of molecular analysis for NF1 and ras alterations in their bone marrow, and summarize their experience with SMNs in pediatric patients with NF1. METHODS Monosomy 7 MDS was diagnosed as an SMN in five children with NF1 by morphologic and cytogenetic studies of bone marrow specimens. DNA extracted from these malignant bone marrows was analyzed for allelic loss at the NF1 locus and for the presence of ras mutations. The Children's Hospital of Philadelphia (CHOP) Tumor Registry was also reviewed to estimate the incidence of SMNs in pediatric NF1 patients. RESULTS Bone marrow specimens from four patients retained constitutional heterozygosity at the NF1 locus; one specimen was uninformative. There was no evidence of activating ras mutations. The risk of an SMN was approximately 11% among the 64 NF1 registrants with primary malignancies in the CHOP registry, but was 75% (6 of 8) among patients treated for a pediatric embryonal cancer. CONCLUSIONS Children with NF1 are susceptible to the development of malignant myeloid disorders both as a primary event and as an SMN. Additional molecular genetic analysis is necessary to determine if the NF1 gene is inactivated by somatic mutation in these secondary leukemias. Cancer 1997; 79:1438-46. © 1997 American Cancer Society.

Patterns of Hematopoietic Lineage Involvement in Children With Neurofibromatosis Type 1 and Malignant Myeloid Disorders

AbstractChildren with neurofibromatosis type 1 (NF1) are at increased risk of developing malignant myeloid disorders, particularly juvenile chronic myelogenous leukemia/juvenile myelomonocytic leukemia (JCML/JMML). We investigated bone marrows from 11 such patients (8 boys and 3 girls) and detected allelic losses at the NF1 locus in 4 of them and probable losses in 2 others. To determine which hematopoietic cell lineages were derived from the abnormal clones, Epstein-Barr virus (EBV)-transformed cell lines and CD34+ cells were analyzed from 3 children with JCML with allelic losses in unfractionated marrow. CD34 cells from these 3 patients lacked the normal NF1 allele, whereas EBV cell lines retained it. Erythroblasts plucked from the burst-forming unit-erythroid colonies of one of these children lacked the normal NF1 allele. We also studied a 10-month-old boy with NF1 who developed an unusual myeloproliferative syndrome. His bone marrow and EBV cell line both showed loss of the normal NF1 allele. In our series and in the literature, male sex and maternal transmission of NF1 were associated with the highest risk of myeloid leukemia. These data (1) provide strong genetic evidence that NF1 functions as a tumor-suppressor in early myelopoiesis, (2) confirm the clonal nature of JCML/JMML, (3) suggest that the elevation in fetal hemoglobin seen in JCML/JMML is a result of primary involvement of erythroid progenitors in the malignant clone, (4) show consistent loss of NF1 in the CD34 cells of affected children and show that the malignant clone may also give rise to pre-B cells in some cases, and (5) implicate epigenetic factors in the development of leukemia in children with NF1.

Patterns of hematopoietic lineage involvement in children with neurofibromatosis type 1 and malignant myeloid disorders

Children with neurofibromatosis type 1 (NF1) are at increased risk of developing malignant myeloid disorders, particularly juvenile chronic myelogenous leukemia/juvenile myelomonocytic leukemia (JCML/JMML). We investigated bone marrows from 11 such patients (8 boys and 3 girls) and detected allelic losses at the NF1 locus in 4 of them and probable losses in 2 others. To determine which hematopoietic cell lineages were derived from the abnormal clones, Epstein-Barr virus (EBV)-transformed cell lines and CD34+ cells were analyzed from 3 children with JCML with allelic losses in unfractionated marrow. CD34 cells from these 3 patients lacked the normal NF1 allele, whereas EBV cell lines retained it. Erythroblasts plucked from the burst-forming unit-erythroid colonies of one of these children lacked the normal NF1 allele. We also studied a 10-month-old boy with NF1 who developed an unusual myeloproliferative syndrome. His bone marrow and EBV cell line both showed loss of the normal NF1 allele. In our series and in the literature, male sex and maternal transmission of NF1 were associated with the highest risk of myeloid leukemia. These data (1) provide strong genetic evidence that NF1 functions as a tumor-suppressor in early myelopoiesis, (2) confirm the clonal nature of JCML/JMML, (3) suggest that the elevation in fetal hemoglobin seen in JCML/JMML is a result of primary involvement of erythroid progenitors in the malignant clone, (4) show consistent loss of NF1 in the CD34 cells of affected children and show that the malignant clone may also give rise to pre-B cells in some cases, and (5) implicate epigenetic factors in the development of leukemia in children with NF1.