Recurrent NF1 gene mutation in a patient with oligosymptomatic neurofibromatosis type 1 (NF1)

We report a 21-year-old male with symptomatic optic glioma who does not fulfill the diagnosis of neurofibromatosis 1 (NF1) according to standard NIH criteria. Analysis of the NF1 gene revealed a recurrent mutation in exon 37 (C6792A or Y2264X). This nonsense mutation causes skipping of exon 37 during the splicing process and is predicted to result in a protein shortened by 34 amino acid residues. The mutation was detected in all tissues examined (blood lymphocytes, oral mucosa, and dermal fibroblasts). The same mutation was previously found in 3 patients with clinically confirmed NF1. To our knowledge, this is the first report of an adult patient carrying a putative (non-mosaic) NF1 gene mutation in multiple tissues but not fulfilling the NIH criteria for the clinical diagnosis of NF1. Am. J. Med. Genet. 86:328–330, 1999.     

Somatic NF1 mutation spectra in a family with neurofibromatosis type 1: toward a theory of genetic modifiers

Neurofibromatosis type 1 (NF1), an autosomal dominantly-inherited disorder, is mainly characterized by the occurrence of multiple dermal neurofibromas and is caused by mutations in the NF1 gene, a tumor suppressor gene. The variable expressivity of the disease and the lack of a genotype/phenotype correlation prevents any prediction of patient outcome and points to the action of genetic factors in addition to stochastic factors modifying the severity of the disease. The analysis of somatic NF1 gene mutations in neurofibromas from NF1 patients revealed that each neurofibroma results from an individual second hit mutation, indicating that factors that influence somatic mutation rates may be regarded as potential modifiers of NF1. A mutational screen of numerous neurofibromas from two NF1 patients presented here revealed a predominance of point mutations, small deletions, and insertions as second hit mutations in both patients. Seven novel mutations are reported. Together with the results of studies that showed LOH as the predominant second hit in neurofibromas of other patients, our results suggest that in different patients different factors may influence the somatic mutation rate and thereby the severity of the disease.     

Comprehensive NF1 screening on cultured Schwann cells from neurofibromas

Neurofibromatosis type 1 (NF1) is mainly characterized by the occurrence of benign peripheral nerve sheath tumors or neurofibromas. Thorough investigation of the somatic mutation spectrum has thus far been hampered by the large size of the NF1 gene and the considerable proportion of NF1 heterozygous cells within the tumors. We developed an improved somatic mutation detection strategy on cultured Schwann cells derived from neurofibromas and investigated 38 tumors from nine NF1 patients. Twenty-nine somatic NF1 lesions were detected which represents the highest NF1 somatic mutation detection rate described so far (76%). Furthermore, our data strongly suggest that the acquired second hit underlies reduced NF1 expression in Schwann cell cultures. Together, these data clearly illustrate that two inactivating NF1 mutations, in a subpopulation of the Schwann cells, are required for neurofibroma formation in NF1 tumorigenesis. The observed somatic mutation spectrum shows that intragenic NF1 mutations (26/29) are most prevalent, particularly frameshift mutations (12/29, 41%). We hypothesize that this mutation signature might reflect slightly reduced DNA repair efficiency as a trigger for NF1 somatic inactivation preceding tumorigenesis. Joint analysis of the current and previously published NF1 mutation data revealed a significant difference in the somatic mutation spectrum in patients with a NF1 microdeletion vs. non-microdeletion patients with respect to the prevalence of loss of heterozygosity events (0/15 vs. 41/81). Differences in somatic inactivation mechanism might therefore exist between NF1 microdeletion patients and the general NF1 population.     

NF1 gene analysis based on DHPLC

The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. The majority of these mutations are private and rare, generating elevated allelic diversity with a restricted number of recurrent mutations. In this study, we have assessed the efficacy of denaturing high-performance liquid chromatography (DHPLC), for detecting mutation in the NF1 gene. DHPLC is a fast and highly sensitive technique based on the detection of heteroduplexes in PCR products by ion pair reverse-phase HPLC under partially denaturing conditions. We established theoretical conditions for DHPLC analysis of all coding exons and splice junctions of the NF1 gene using the WAVEmaker software version 4.1.40 and screened for mutations a panel of 40 unrelated NF1 patients (25 sporadic and 15 familial), genetically uncharacterized. Disruptive mutations were identified in 29 individuals with an overall mutation detection rate of 72.5%. The mutations included eight deletions (exons 4b, 7, 10a, 14, 26, and 31), one insertion (exon 8), nine nonsense mutation (exons 10a, 13, 23.1, 27a, 29, 31, and 36), six missense mutations (exons 15, 16, 17, 24, and 31), four splice errors (exons 11, 14, 36, and 40) and a complex rearrangement within exon 16. Eighteen (62%) of the identified disruptive mutations are novel. Seven unclassified and three previously reported polymorphisms were also detected. None of the missense mutations identified in this study were found after screening of 150 controls. Our results suggest that DHPLC provides an accurate method for the rapid identification of NF1 mutations.     

A novel mutation L1425p in the GAP‐region of the NF1 gene detected by temperature gradient gel electrophoresis (TGGE);

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with an incidence of between 1: 3000 and 1: 4000. Common clinical signs include more than six café‐au‐lait spots, multiple cutaneous neurofibromas and iris Lisch nodules. Rarer are skeletal anomalies, learning disabilities and an increased risk of malignancy. The NF1 gene contains at least 60 exons with intron sizes ranging from 60 bp to more than 40 kb. Despite using different techniques including PTT, SSCP, heteroduplex analyses and direct sequencing, only a relatively small number of mutations have been reported world‐wide. Using the more sensitive technique of temperature gradient gel electrophoresis (TGGE), we analysed a part of the NF1‐GAP‐region, namely exon 25, in DNA samples from 131 unrelated patients. We have identified a novel mutation L1425P in exon 25 of the NF1 gene in a 12‐year‐old boy (clinically diagnosed with NF1 at the age of 7). In contrast to those cases diagnosed with having both GAP‐region mutations and malignant tumours, neither the proband nor four clinically affected family members with this mutation showed any evidence of malignancies. © 1999 Wiley‐Liss, Inc.     

Noonan syndrome and neurofibromatosis type I in a family with a novel mutation in NF1

Noonan syndrome (NS) and neurofibromatosis type I (NF1) belong to a group of clinically related disorders that share a common pathogenesis, dysregulation of the RAS‐MAPK pathway. NS is characterized by short stature, heart defect, pectus deformity and facial dysmorphism, whereas skin manifestations, skeletal defects, Lisch nodules and neurofibromas are characteristic of NF1. Both disorders display considerable clinical variability. Features of NS have been observed in individuals with NF1 –a condition known as neurofibromatosis–Noonan syndrome (NFNS). The major gene causing NFNS is NF1. Rarely, a mutation in PTPN11 in addition to an NF1 mutation is present. We present the clinical and molecular characterization of a family displaying features of both NS and NF1, with complete absence of neurofibromas. To investigate the etiology of the phenotype, mutational analysis of NF1 was conducted, revealing a novel missense mutation in exon 24, p.L1390F, affecting the GAP‐domain. Additional RAS‐MAPK pathway genes were examined, but no additional mutations were identified. We confirm that NF1 mutations are involved in the etiology of NFNS. Furthermore, based on our results and previous studies we suggest that evaluation of the GAP‐domain of NF1 should be prioritized in NFNS.     

Unusual clustering of brain tumours in a family with NF1 and variable expression of cutaneous features

Neurofibromatosis type 1 (NF1) is one of the commonest autosomal dominant disorders in man. It is characterised by café au lait spots, peripheral neurofibromas, Lisch nodules, axillary freckling, skeletal dysplasia, and optic glioma. Symptomatic brain tumours occur in 1.5-2.2% of patients with NF1. We report here a family where seven members developed brain tumours. Of these, three have a clinical history strongly suggestive of NF1, while two do not fulfil diagnostic criteria for the disorder. A splice site mutation in exon 29 of the NF1 gene was found in these two subjects. This lesion is thought to be disease causative since it creates a frameshift and a premature termination of the neurofibromin protein. Different hypotheses to explain the unusual recurrence of brain tumours in this family, such as the nature of the mutation or cosegregation of other predisposing genes, are discussed.     

Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1)

Mosaicism is an important feature of type-1 neurofibromatosis (NF1) on account of its impact upon both clinical manifestations and transmission risk. Using FISH and MLPA to screen 3500 NF1 patients, we identified 146 individuals harboring gross NF1 deletions, 14 of whom (9.6%) displayed somatic mosaicism. The high rate of mosaicism in patients with NF1 deletions supports the postulated idea of a direct relationship between the high new mutation rate in this cancer predisposition syndrome and the frequency of mosaicism. Seven of the 14 mosaic NF1 deletions were type-2, whereas four were putatively type-1, and three were atypical. Two of the four probable type-1 deletions were confirmed as such by breakpoint-spanning PCR or SNP analysis. Both deletions were associated with a generalized manifestation of NF1. Independently, we identified a third patient with a mosaic type-1 NF1 deletion who exhibited segmental NF1. Together, these three cases constitute the first proven mosaic type-1 deletions so far reported. In two of these three mosaic type-1 deletions, the breakpoints were located within PRS1 and PRS2, previously identified as hotspots for nonallelic homologous recombination (NAHR) during meiosis. Hence, NAHR within PRS1 and PRS2 is not confined to meiosis but may also occur during postzygotic mitotic cell cycles.     

Motor deficits and neurofibromatosis type 1 (NF1)‐associated MRI impairments in a mouse model of NF1

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized inter alia by cognitive and motor dysfunction and appearance of high‐signal foci on T2‐weighted images in the brain. Nf1^+/? mice are useful models for studying aspects of NF1, including cognitive deficits. Here we assessed their motor performance and used quantitative transverse T2 relaxation MRI to identify structural abnormalities in their brains. Nf1^+/? mice exhibited both enhanced and reduced T2 signals in distinct brain regions compared to wild‐type mice, and their motor performance was impaired. As in NF1 patients, enhanced T2 signals in Nf1^+/? mice were observed in the thalamus and basal ganglia. Reduced T2 signals were seen in motor‐associated regions along the motor pathway, predominantly in the white matter of the cerebral peduncle and the optic tract. Correlation analysis between T2 signals and motor performance suggested that the motor deficits are associated with impairments in the cerebral peduncle and the amygdala. Copyright © 2010 John Wiley & Sons, Ltd.     

Ovarian cancer BRCA1 mutation detection: Protein truncation test (PTT) outperforms single strand conformation polymorphism analysis (SSCP)

Recent studies have shown that the BRCA1 tumor suppressor gene plays a role in the development of both hereditary and sporadic ovarian cancer. Since several different mechanisms may give rise to tumor gene defects, a better understanding of these mechanisms may identify BRCA1 as an attractive therapeutic target in ovarian cancer. Sequencing this large gene is not practical on a population‐wide basis. The optimal screening strategy is yet to be determined. The purpose of our study is to compare two common screening techniques: the protein truncation test (PTT) and single strand conformational polymorphism analysis (SSCP). Ninety‐four patients with epithelial ovarian cancer and available snap‐frozen tissue were screened for BRCA1 mutations by both PTT (five individual PCR reactions with complete translation of the product in the TNT System (Promega, Madison, WI)) and SSCP (41 individual PCR reactions covering the entire coding sequence). All abnormal results were confirmed by sequencing. A paired peripheral blood DNA sample was utilized to determine if the sequence abnormality was a germline mutation. Twenty‐three mutations in BRCA1 were found in 22 patients (14 germline, eight somatic, one unknown) including four novel mutations: E489X, 3558delT, 3871delGTCT, del exon 7–10. Although the predictive value of a negative test was close for the two methods (PTT 99.1%, SSCP 99.8%), the comparison of positive predictive value overwhelmingly favored PTT (100.0%, vs. 26.4%, respectively). The specificity for PTT was 100.0% while the sensitivity was 82.6%. While for SSCP, the specificity was 99.0% and the sensitivity was only 60.9%. The concordance rate for the two screening tests was 88.9%. Only SSCP can detect missense mutations. PTT is a superior screening test for truncating BRCA1 mutations that are expected to be of clinical significance. Hum Mutat 18:337–344, 2001. © 2001 Wiley‐Liss, Inc.     

Academic Impairment is the Most Frequent Complication of Neurofibromatosis Type-1 (NF1) in Children

Neurofibromatosis type-1 (NF1) is a common genetic disorder associated with a variety of medical complications, cognitive impairments, and behavioral problems. One hundred and sixteen patients with NF1 (62 males, 54 females; mean age 12.4 years) and 80 typically developing children of the same ages (46 males, 34 females; mean age 11.5 years) were studied in terms of complications and learning impairment (one or more grade repetitions or school exclusion). Seventy of 116 patients had significant learning impairment. Classical complications were present in 53 patients including the three.     

Genotype analysis of the NF1 gene in the French Canadians from the Québec population

We genotyped 19 NF1 families from the French Canadians of the Québec population with six intragenic polymorphic markers including 2 RFLPs (EcoRI and RsaI) and 4 microsatellites (IVS26‐2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Genotype analysis indicated families 7610 and 7473 bear deletions. In Family 7610 the deletion removed the entire NF1 gene except exons 1 to 4b. The breakpoint of the deletion is located between exons 4a and 4b. The deletion 7473 was derived from the maternal chromosome and exons 1 to 5 were deleted. The breakpoint of the deletion is located between exons 7 and 13. Their phenotypes are reported. The allele frequencies of microsatellites IVS27AC28.4 and IVS38GT53.0 are compared to previously reported data from Caucasians, including Spanish and Italians. The difference is statistically significant (P < 0.0036) for marker IVS27AC28.4 between the Québec French Canadian and the Italian population. © 2001 Wiley‐Liss, Inc.     

Multiple spinal ganglioneuromas in a patient harboring a pathogenic NF1 mutation

Bacci C, Sestini R, Ammannati F, Bianchini E, Palladino T, Carella M, Melchionda S, Zelante L, Papi L. Multiple spinal ganglioneuromas in a patient harboring a pathogenic NF1 mutation. Ganglioneuroma is a rare benign tumor originating from autonomic ganglia and is considered the benign counterpart of neuroblastoma. Ganglioneuromas may be present as an isolated finding and, rarely, in association with neurofibromatosis type 1 (NF1). However, ganglioneuromas of the cervical spine with intradural extension and multiple locations are extremely rare. We describe a 32‐year‐old woman with multiple ganglioneuromas of the cervical, dorsal and lumbar spine associated with a few café‐au‐lait spots and subcutaneous nodules. The patient lacked other NF1 stigmata, such as freckling, Lisch nodules and cutaneous neurofibromas. Although our patient did not fulfill the NF1 diagnostic criteria, molecular diagnosis revealed a pathogenic mutation in the NF1 gene. Approximately 30 patients affected by NF1 and ganglioneuromas have been reported: in all these individuals, NF1 diagnosis was made according to the clinical diagnostic criteria and no patients have molecular diagnosis. Therefore, this is the first case with multiple spinal ganglioneuromas associated with a pathogenic NF1 mutation.     

Null phenotype of neurofibromatosis type 1 in a carrier of a heterozygous atypical NF1 deletion due to mosaicism

We coincidently detected an atypical deletion of at least 1.3‐Mb, encompassing the NF1 tumor suppressor gene and several adjacent genes at an apparent heterozygous level in the blood of a 65‐year‐old female patient. She had multiple subcutaneous tumors that appeared with a certain similarity of subcutaneous neurofibromas, which, however, was revealed as lipomas by histological examination. Comprehensive and exhaustive clinical and radiological examinations did not detect any neurofibromatosis type 1‐related clinical symptoms in the patient. Multiplex ligation‐dependent probe amplification detected no or only very low level of the 1.3‐Mb NF1 deletion in six lipomas and two skin biopsies. Digital polymerase chain reaction estimated the proportion of cells carrying a heterozygous NF1 deletion at 87% in the blood, and 8%, 10%, 13%, 17%, and 20%, respectively, in the five lipomas investigated by this method, confirming our hypothesis of mosaicism. Our findings suggest that de novo cases of genetic disease are potentially mosaic regardless of finding the mutation at an apparently heterozygous level in the blood and that the possibility of mosaicism should be considered in genotype–phenotype studies and genetic counseling.     

Germline and somatic NF1 gene mutation spectrum in NF1-associated malignant peripheral nerve sheath tumors (MPNSTs)

About 10% of neurofibromatosis type 1 (NF1) patients develop malignant peripheral nerve sheath tumors (MPNSTs) and represent considerable patient morbidity and mortality. Elucidation of the genetic mechanisms by which inherited and acquired NF1 disease gene variants lead to MPNST development is important. A study was undertaken to identify the constitutional and somatic NF1 mutations in 34 MPNSTs from 27 NF1 patients. The NF1 germline mutations identified in 22 lymphocytes DNA from these patients included seven novel mutations and a large 1.4-Mb deletion. The NF1 germline mutation spectrum was similar to that previously identified in adult NF1 patients without MPNST. Somatic NF1 mutations were identified in tumor DNA from 31 out of 34 MPNSTs, of which 28 were large genomic deletions. The high prevalence (>90%) of such deletions in MPNST contrast with the =or<20% found in benign neurofibromas and is indicative of the involvement of different mutational mechanisms in these tumors. Coinactivation of the TP53 gene by deletion, or by point mutation along with NF1 gene inactivation, is known to exacerbate disease symptoms in NF1, therefore TP53 gene inactivation was screened. DNA from 20 tumors showed evidence for loss of heterozygosity (LOH) across the TP53 region in 11 samples, with novel TP53 point mutations in four tumors.     

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).     

一例散发1型神经纤维瘤病患者的NF1基因嵌合突变分析

目的 对1例散发1型神经纤维瘤病患者的NF1基因进行嵌合突变分析.方法 提取先证者外周血基因组RNA,PCR扩增NF1基因编码区序列并进行序列测定;找到突变后,基因组DNA途径证实突变,并对先证者儿子的NF1基因相应外显子也进行序列分析;针对NF1基因第51外显子已发现的突变,取先证者全血淋巴细胞、口腔上皮细胞和尿路上皮细胞基因组DNA进行PCR扩增,PCR产物T克隆及测序.结果 先证者的临床表现符合1型神经纤维瘤病.先证者外周血RNA途径检测出无义突变c.7911C＞T(p.Q2510X);基因组DNA途径证实患者外周血淋巴细胞、口腔上皮细胞和尿路上皮细胞中均有该突变,尿路上皮细胞中该突变测序信号较弱;在PCR产物的T克隆-测序中,来自先证者的血液、口腔上皮、尿液上皮细胞无义突变c.7911C＞T(p.Q2510X)突变体的重组菌分别占总数的42％、36％、12％.其儿子、正常对照不存在上述突变.结论 先证者在胚胎早期发生了NF1基因突变,使其体内部分细胞带有NF1基因突变,导致形成全身嵌合的1型神经纤维瘤病.     

NF1基因新突变及我国首例NF1基因拷贝数目变异报道

 目的:对2例散发性1型神经纤维瘤患者进行致病基因NF1编码序列的突变筛查以及拷贝数变异(copy number variation,CNV)研究,寻找致病性突变。方法:PCR扩增NF1基因的编码区及外显子-内含子交界区,对产物进行直接测序。在50例正常对照中进行新发现突变位点的测序分析,以排除多态性。用多重连接探针扩增技术(MLPA)对患者进行NF1基因CNV的检测,并对证实有NF1拷贝缺失的患者进行长片段PCR,以找寻断裂点。结果:患者S736于NF1基因检测到一个国际上尚未报道的新突变：c.6345_6346 ins G (p.Leu2116Alafs*4),患者父母均不携带此突变,故此突变为一个de novo突变。该突变使开放阅读框移位,提前引入终止密码子,导致蛋白质分子的截断,以致部分犰狳式褶皱(ARM-type fold)结构域丢失。在另一患者S743中检测到一个包含整个NF1基因的大片段缺失,缺失区域为1.3～1.9 Mb,但断裂点尚不明,此为我国首例NF1基因拷贝数目变异的报道。结论: 患者NF1基因的de novo突变及CNV是引起这2例1型神经纤维瘤发病的分子机制,这些发现可用于临床1型神经纤维瘤的分子诊断。