Fast and Robust Next-Generation Sequencing Technique Using Ion Torrent Personal Genome Machine for the Screening of Neurofibromatosis Type 1 (NF1) Gene

Neurofibromatosis type 1 (NF1) gene exhibits one of the highest spontaneous mutation rates in the human genome. Identification of the NF1 mutation is challenging because the NF1 gene is very large and complex, lacking mutational “hot spots.” There is no clustering of mutations, there are several pseudogenes, and a wide spectrum of different types of mutation has been recognized. To date, NF1 mutations or deleted regions have been detected with a number of techniques. With the appearance of next-generation sequencing (NGS) machines, molecular biology is in a new revolutionary phase. Our aim was to work out a method to use the high-throughput NGS machine, Ion Torrent PGM, in diagnostic settings for neurofibromatosis type 1. In our examination, we could reveal 21 distinct variations in NF1 gene in seven patients. This is an absolutely new method for exploring the genetic background of neurofibromatosis type 1 exhibiting the extremely high throughput of NGS in a diagnostic setting.     

A novel mutation in NF1 is associated with diverse intra-familial phenotypic variation and astrocytoma in a Chinese family

Neurofibromatosis type 1 (NF1) is a dysregulated neurocutaneous disorder, characterized by neurofibromas and café-au-lait spots. NF1 is caused by mutations in the NF1 gene, encoding neurofibromin. Here, we present a clinical molecular study of a three-generation Chinese family with NF1. The proband was a male patient who showed café-au-lait spots and multiple subcutaneous neurofibromas over the whole body, but his siblings only had regional lesions. The man’s daughter presented with severe headache and vomiting. Neurological examination revealed an intracranial space occupying lesion. Surgery was undertaken and the histopathological examination showed a grade I-II astrocytoma. Next-Generation sequencing (Illumina HiSeq2500 Analyzers; Illumina, San Diego, CA, USA) and Sanger sequencing (ABI PRISM 3730 automated sequencer; Applied Biosystems, Foster City, CA, USA) identified the c.227delA mutation in the NF1 gene in the man. The mutation is co-segregated with the disease phenotypes among the affected members of this family and was absent in 100 healthy controls. This novel mutation results in a frameshift (p.Asn78IlefsX7) as well as truncation of neurofibromin by formation of a premature stop codon. Our results not only extended the mutational and phenotypic spectra of the gene and the disease, but also highlight the importance of the other genetic or environmental factors in the development and severity of the disease.     

Cerebral vasculopathy in a Chinese family with neurofibromatosis type I mutation

Neurofibromatosis type I (NF1) is a hereditary, autosomal dominant, neurocutaneous syndrome that is attributed to NF1 gene mutation. NF1 has been associated with scoliosis, macrocephaly, pseudoarthrosis, short stature, mental retardation, and malignancies. NF1-associated vasculopathy is an uncommon and easily-overlooked presentation. Examination of a Chinese family affected by NF1 combined with cerebral vessel stenosis and/or abnormality suggested a possible relationship between NF1 and vessel stenosis. To determine which NF1 gene mutation is associated with vascular lesions, particularly cerebral vessel stenosis, we examined one rare family with combined cerebral vessel lesions or maldevelopment. Vascular lesions were detected using transcranial Doppler sonography and digital subtraction angiography in family members. Next, denaturing high-performance liquid chromatography and sequencing were used to screen for NF1 gene mutations. The results revealed a nonsense mutation, c.541C>T, in the NF1 gene. This mutation truncated the NF1 protein by 2659 aminoacid residues at the C-terminus and co-segregated with all of the patients, but was not present in unaffected individuals in the family. Exceptionally, three novel mutations were identified in unaffected family members, but these did not affect the product of the NF1 gene. Thus the nonsense mutation, c.541C>T, located in the NF1 gene could constitute one genetic factor for cerebral vessel lesions.     

Familial spinal neurofibromatosis: clinical and DNA linkage analysis.

We studied two families with an unusual variant of neurofibromatosis (NF). The first family had spinal neurofibromas and café au lait spots (CLS), the second spinal neurofibromas without CLS. Other signs of NF1 or NF2, such as cutaneous tumors, Lisch nodules, or acoustic tumors, were absent. The inheritance pattern in both pedigrees was consistent with autosomal dominant inheritance. Using genetic linkage analysis with DNA markers tightly linked to the NF1 and NF2 loci, we determined that the likely location for the mutation in the first family was in the NF1 gene with odds of 97:1, whereas the mutation in the second family was excluded from the NF1 locus with odds greater than 100,000:1. Families such as these, in which a defined subset of the NF phenotype is passed on, are important for understanding the functional consequences of particular mutations in the NF genes.     

Spinal neurofibromatosis in a family with classical neurofibromatosis type 1 and a novel NF1 gene mutation

Familial spinal neurofibromatosis (FSNF) is a rare form of neurofibromatosis type 1 (NF1) characterized by multiple, histologically proven neurofibromas of the spinal roots leaving no intact segments and associated neurofibromas of major peripheral nerves. It is sometimes associated with other NF1 stigmata. Most patients have NF1 gene mutations. We describe a patient who fulfilled the diagnostic criteria for spinal neurofibromatosis and belonged to a family in which other affected members exhibited classical NF1 stigmata. A novel missense (c.7109 T > A; p.Val2370Asp) mutation in exon 39 of the NF1 gene was present in the affected family members. The family displayed extreme phenotypic variability in the spectrum of NF1. To our knowledge, this is the first patient with spinal neurofibromatosis in the context of classical NF1 with an NF1 gene mutation. The term FSNF is inaccurate as this condition simply reflects the typical autosomal dominant pattern of NF1 inheritance with phenotypoc variability and does not encompass patients with sporadic disease or those in the context of a classical NF1 phenotype as reported in the present family. The term could be replaced by “spinal neurofibromatosis”.     

神经纤维瘤病1型基因32、33号外显子突变的检测

目的检测中国人神经纤维瘤病１型（ＮＦ１）基因３２、３３外显子突变。方法应用聚合酶链反应－单链构象多态性（ＰＣＲＳＳＣＰ）法分析１４个ＮＦ１家系６２名成员及３０名正常对照外周血白细胞ＤＮＡ的ＮＦ１基因３２、３３号外显子。结果３个家系（２１．４％）、４例ＮＦ１患者（１１．１％）ＮＦ１基因３２号外显子的ＤＮＡＳＳＣＰ发生泳动变位,可能为ＮＦ１基因３２号外显子发生突变。家系中其他成员及正常对照无此现象。通过３种不同的ＳＳＣＰ实验条件,未发现３３号外显子异常泳动变位。结论３２号外显子可能为中国人ＮＦ１基因突变热点之一。本研究方法对ＮＦ１症状前诊断和产前诊断有重要应用价值。     

Diagnosis of neurofibromatosis type 1 using RFLPs tightly linked to gene

This study reports the results of a linkage analysis in nine families with members who had neurofibromatosis type 1 (NF1), using five restriction fragment length polymorphisms (RFLPs) tightly linked to the NF1 locus. The purpose of this analysis was to determine whether the at-risk individuals were carrying the NF1 allele and whether the nine families would be informative for prenatal testing. The families included 25 patients with NF1, 3 individuals at risk for NF1, and 11 unaffected subjects, with a total of 39 family members and 12 matings. In 6 matings two or more flanking probes were informative, in 3 matings only one probe was informative, and in the other 3 no probe was informative. DNA linkage analysis showed with more than 98% probability that the 3 at-risk individuals did not carry the NF1 mutation. No recombination events were observed. In 6 families it will be possible to do a DNA prenatal diagnosis if this type of test is requested. The NF1 gene has been identified and direct testing for the NF1 mutation is now possible. Linkage testing, however, will probably remain useful and complementary to direct analysis of the NF1 gene to reveal intragenic recombination events and for diagnosis in families in which the detection of mutation is difficult.     

Ⅰ型神经纤维瘤病基因GRD区突变研究

目的 对39例Ⅰ型神经纤维瘤病（NF1）患者基因的部分GTP酶活化蛋白相关功能区（GRD区）的cDNA突变进行研究。方法 应用反转录聚合酶链反应（RT-PCR）分段扩增GRD区域上的cDNA序列,用单链构象多态性（SSCP）及直接测序法对RT-PCR产物进行突变分析。结果 仅发现1例点突变G3918T颠换,导致1306精氨酸（R）变成亮氨酸（L）。结论NF1基因的GRD区可能不是NF1基因突变的热点区域。     

How does the Schwann cell lineage form tumors in NF1?

Neurofibromas are benign tumors of peripheral nerve that occur sporadically or in patients with the autosomal dominant tumor predisposition syndrome neurofibromatosis type 1 (NF1). Multiple neurofibroma subtypes exist which differ in their site of occurrence, their association with NF1, and their tendency to undergo transformation to become malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with NF1. Most NF1 patients carry a constitutional mutation of the NF1 tumor suppressor gene. Neurofibromas develop in these patients when an unknown cell type in the Schwann cell lineage loses its remaining functional NF1 gene and initiates a complex series of interactions with other cell types; these interactions may be influenced by aberrant expression of growth factors and growth factor receptors and the action of modifier genes. Cells within certain neurofibroma subtypes subsequently accumulate additional mutations affecting the p19(ARF)-MDM2-TP53 and p16INK4A-Rb signaling cascades, mutations of other as yet unidentified genes, and amplification of growth factor receptor genes, resulting in their transformation into MPNSTs. These observations have been validated using a variety of transgenic and knockout mouse models that recapitulate neurofibroma and MPNST pathogenesis. A new generation of mouse models is also providing important new insights into the identity of the cell type in the Schwann cell lineage that gives rise to neurofibromas. Our improving understanding of the mechanisms underlying the pathogenesis of neurofibromas and MPNSTs raises intriguing new questions about the origin and pathogenesis of these neoplasms and establishes models for the development of new therapies targeting these neoplasms.     

Ⅰ型神经纤维瘤病基因28及31号外显子突变的检测

目的　探讨国人Ⅰ型神经纤维瘤病 (NF1)基因突变的热点。方法　用PCR SSCP方法检查NF1基因 2 8和 3 1号外显子共约 10 60bp ,约占NF1基因全部编码区的 6 95 %。结果　在 2 7个家系中 ,发现 4个家系 (14 82 % )、13例病人(2 3 64 % )存在NF1基因突变。结论　提示本组病例 2 8和 3 1号外显子可能为突变热点。对突变性质的定论有赖于DNA序列分析。     

A novel NF1 gene mutation in an Italian family with neurofibromatosis type 1

Purpose

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder with an estimated incidence of one in 3,500 births. Clinically, NF1 is characterized by café-au-lait (CAL) spots, neurofibromas, freckling of the axillary or inguinal region, Lisch nodules, optic nerve glioma, and bone dysplasias. NF1 is caused by inactivating mutations of the 17q11.2-located NF1 gene. We present a clinical and molecular study of an Italian family with NF1.

Methods

The proband, a 10-year-old boy, showed large CAL spots and freckling on the axillary region and plexiform neurofibromas on the right side only. His father (47?years old) showed, in addition to the similar signs, numerous neurofibromas of various sizes on his thorax, abdomen, back, and shoulder. Two additional family members (a brother and a sister of the proband) presented only small CAL spots. The coding exons of NF1 gene were analyzed for mutations by denaturing high-performance liquid chromatography and sequencing in all family members.

Results

The mutational analysis of the NF1 gene revealed a novel frameshift insertion mutation in exon 4c (c.654 ins A) in all affected family members. This novel mutation creates a shift on the reading frame starting at codon 218 and leads to the introduction of a premature stop at codon 227.

Conclusions

The segregation of the mutation with the affected phenotype and its absence in the 200 normal chromosomes suggest that it is responsible for the NF1 phenotype.     

A new nonsense mutation in the NF1 gene with neurofibromatosis–Noonan syndrome phenotype

Purpose

Neurofibromatosis–Noonan syndrome is a rare autosomal dominant disorder which combines neurofibromatosis type 1 (NF1) features with Noonan syndrome. NF1 gene mutations are reported in the majority of these patients.

Method

Sequence analysis of the established genes for Noonan syndrome revealed no mutation; a heterozygous NF1 point mutation c.7549C>T in exon 51, creating a premature stop codon (p.R2517X), had been demonstrated.

Result

Neurofibromatosis–Noonan syndrome recently has been considered a subtype of NF1 and caused by different NF1 mutations.

Conclusion

We report the case of a 14-year-old boy with neurofibromatosis type 1 with Noonan-like features, who complained of headache with triventricular hydrocephaly and a heterozygous NF1 point mutation c.7549C>T in exon 51.     

Detailed analysis of the oligodendrocyte myelin glycoprotein gene in four patients with neurofibromatosis 1 and primary progressive multiple sclerosis

Neurofibromatosis 1 (NF1) is a common autosomal disorder with a wide range of neurological manifestations. The case histories of five patients, including two siblings, are reported who have both neurofibromatosis 1 and primary progressive multiple sclerosis (PPMS). A further patient with both NF1 and PPMS has since been identified. More recently, a systematic clinical review of 138 unselected adult patients with NF1 identified one patient with a slowly progressive spastic paraparesis and multiple high signal hyperintensities on T2 weighted MRI. Molecular genetic studies suggest a mechanism by which the clinical association of progressive white matter disease and NF1 might arise. The gene for NF1 is located on chromosome 17q, spans 350 kb of genomic DNA, and contains 60 exons. The gene for oligodendrocyte myelin glycoprotein (OMgp) is embedded within intron 27b of the NF1 gene. OMgp is a membrane glycoprotein that appears in the human CNS at the time of myelination. It can be detected immunohistochemically on CNS myelin and on the surface of cultured oligodendrocytes. Structurally, OMgp has the potential to function as an adhesion molecule and could contribute to the interactions between the plasma membranes of oligodendrocytes and axons required for myelination and/or axon survival. This study considers the specific hypothesis that PPMS in patients with NF1 results from concurrent mutation of the OMgp gene. The OMgp genes of four unrelated patients with NF1 and PPMS were examined using a combination of Southern blot, dosage polymerase chain reaction, and chemical cleavage of mismatch. The entire OMgp coding sequence, all intronic sequence, the intron-exon boundaries, and 1 kb of flanking sequence were screened. The DNA from two patients was found to contain an alteration in the OMgp gene resulting in an amino acid change of glycine to aspartic acid at codon 21. It is concluded that PPMS in patients with NF1 can occur without concurrent mutation of the OMgp gene. The glycine to aspartic acid polymorphic alteration at codon 21 is neither sufficient nor necessary for the development of PPMS.     

Clinicopathologic and genetic analysis of siblings with NF1 and adult-onset gliomas

BACKGROUND: Neurofibromatosis Type 1 (NF1) is a common autosomal dominant neurogenetic disorder characterized by neoplasms involving the nervous system which typically present in children. The development of intracranial tumors in adults with NF1 is uncommon and to our knowledge, siblings with adult onset gliomas have not been previously reported. OBJECTIVE: To perform pathological, clinical and genetic analysis of an unusual family with NF1 and adult onset intracranial gliomas. RESULTS: A 39-year-old woman presented with seizures and aphasia and was diagnosed with an intracerebral tumor. Although there was no family history, she met the accepted clinical criteria for NF1. A biopsy was performed and pathological examination revealed an anaplastic pleomorphic xanthoastrocytoma (PXA). In spite of therapy, she died from complications of tumor recurrence. Her 32-year-old sister developed headaches and was diagnosed with a glioma. Although she did not meet the accepted clinical criteria for NF1, given that she has a sibling with NF1 and a malignancy observed in this disorder, we hypothesize that she also has NF1. Our genetic analysis indicated a shared haplotype in these siblings who developed brain tumors but not in an unaffected sister suggesting that both carry the NF1 disease-producing allele. This haplotype was inherited from their unaffected father indicating a paternal origin of the spontaneous putative mutation in the NF1 gene in this family. CONCLUSION: NF1 should be a diagnostic consideration when siblings develop intracranial brain tumors even when they develop in adults. Our study supports and extends other reports that broaden the clinical and pathological spectrum of manifestations that can occur in NF1 to include not only adult-onset gliomas but uncommon histological subtypes such as PXA.     

Recent developments in neurofibromatosis type 1

PURPOSE OF REVIEW: This review summarizes the recent clinical and genetic developments in neurofibromatosis type 1 (NF1) and provides an insight into the possible underlying pathomechanisms. RECENT FINDINGS: NF1, or von Recklinghausen disease, is one of the most common hereditary neurocutaneous disorders in humans. Clinically, NF1 is characterized by café-au-lait spots, freckling, skin neurofibroma, plexiform neurofibroma, bony defects, Lisch nodules and tumors of the central nervous system. The responsible gene, NF1, encodes a 2818 amino acid protein (neurofibromin). Pathological mutations range from single nucleotide substitutions to large-scale genomic deletions dispersed throughout the gene. In addition to the conventional mutation screening methods, a DNA chip microarray-based technology, combinational sequence-based hybridization, has been introduced to expedite mutation detection. Functional analysis has become more amenable following the development of the following: (1) primary Schwann cell cultures from NF1 patients; (2) mouse models; (3) proteomic technologies; and (4) mRNA silencing by RNA interference. These studies have shown that neurofibromin plays a role in adenylate cyclase and AKT-mTOR mediated pathways. It also appears to affect Ras-GTPase activating protein activity through the phosphorylation of protein kinase C which impacts on cell motility by binding with actin in the cytoskeleton. SUMMARY: Recent advances in the clinical features and molecular genetics of NF1 will be discussed together with insights into the underlying pathomechanisms of NF1.     

NF1 mutations and molecular testing

Neurofibromatosis 1 is a progressive autosomal dominant condition caused by mutations in the NF1 gene on chromosome 17. The condition shows clinical variable expressivity, with varying features even between family members who share the same mutation. Furthermore, it is impossible to precisely predict the severity and course of the condition, a source of frustration for families and physicians. Neurofibromatosis 1 is also heterogeneous at the mutation level, with more than 300 independent mutations having been reported in this gene. The mutation data have accumulated slowly owing to the variability of the mutation types and the size and complexity of the gene. This is also reflected in the lack of a simple, inexpensive, highly accurate DNA-based test for neurofibromatosis 1 at present. This article reviews current NF1 mutation spectrum and testing, discussing and illustrating mutation mechanisms and pathogenetic effects, as well as factors affecting DNA testing and interpretation/diagnosis.     

Seizure, spinal schwannoma, peripheral neuropathy and pulmonary stenosis �C A rare combination in a patient of Neurofibromatosis 1

Neurofibromatosis 1 (NF1) is the most common neurocutaneous syndrome. It is estimated to occur in approximately 1 out of every 3300 infants. The manifestations of this condition are diverse and can arise from almost any system in the body. The neurofibroma is the hallmark lesion of NF1 that develops from peripheral nerves. Here, we are reporting an 18-year-old girl with NF1. Clinical diagnosis was made according to the diagnostic criteria established by the National Institutes of Health Consensus Development Conference in 1987. She presented with quadriparesis due to dumbbell-shaped spinal schwannoma in the cervical region. She had history of recurrent seizures in the past, with poor scholastic performance. There were clinical and electrophysiological features of peripheral neuropathy and clinical and echocardiographical features of pulmonary stenosis. These are uncommon features of NF 1. The presence of all these features in a single patient makes it a unique case.     

Absence of exon 17 c.2970-2872delAAT mutation in Turkish NF1 patients with mild phenotype

Introduction  

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by café-au-lait spots, neurofibromas, skinfold freckles, Lisch nodules, bone deformities, learning disabilities, and predisposition to neoplasms. It is caused by various mutations of the NF1 gene. Recently a 3-bp in-frame deletion in exon 17, c.2970-2972 delAAT mutation, has been associated with a milder phenotype of NF1 manifesting with pigmentary skin changes only.     

Novel mutations in the NF1 gene in Czech patients with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is one of the most common inherited human disorders, with an estimated incidence of 1 per 3500 births. In most cases, the disease is caused either by mutation in the NF1 gene, or by a particular or complete deletion of the NF1 gene. The NF1 gene exhibits one of the highest mutation rates of any human disorder. In this experimental study of the NF1 gene, we screened the mutational spectrum of 22 unrelated patients from the Czech Republic using the denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA) methods. We found NF1 mutations in 17 patients: 15 causal mutations were detected with the use of the DHPLC method (15/20, 75%). With the MPLA method, we also confirmed and specified two large deletions that were previously genotyped by microsatellite markers. Twelve of the above-mentioned mutations were newly found: c.1_2delATinsCC, c.1185+1G>C, c.1757_1760delCTAG, c.1642-7A>G, c.2329 T>G, c.2816delA, c.3738_3741delGTTT, c.4733 C>T, c.5220delT, c.6473_6474insGAAG, ex14_49del, ex28_49del. We present this study as a first effectual step in the routine diagnosis of the NF1 in patients from the Czech Republic.     

Neurofibromatosis 2 and malignant mesothelioma

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene cause the inherited disorder NF2 and are also common in malignant mesothelioma, which is not a characteristic feature of NF2. The authors report an asbestos-exposed person with NF2 and malignant mesothelioma. Immunohistochemical analysis of the mesothelioma confirmed loss of expression of the NF2 protein, and comparative genomic hybridization revealed losses of chromosomes 14, 15, and 22, and gain of 7. The authors propose that a person with a constitutional mutation of an NF2 allele is more susceptible to mesothelioma.