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.     

Cardiac characterization of 16 patients with large NF1 gene deletions

The aim of this study was to characterize cardiac features of patients with neurofibromatosis 1 (NF1) and large deletions of the NF1 gene region. The study participants were 16 patients with large NF1 deletions and 16 age‐ and sex‐matched NF1 patients without such deletions. All the patients were comprehensively characterized clinically and by echocardiography. Six of 16 NF1 deletion patients but none of 16 non‐deletion NF1 patients have major cardiac abnormalities (p = 0.041). Congenital heart defects (CHDs) include mitral insufficiency in two patients and ventricular septal defect, aortic stenosis, and aortic insufficiency in one patient each. Three deletion patients have hypertrophic cardiomyopathy. Two patients have intracardiac tumors. NF1 patients without large deletions have increased left ventricular (LV) diastolic posterior wall thickness (p < 0.001) and increased intraventricular diastolic septal thickness (p = 0.001) compared with a healthy reference population without NF1, suggestive of eccentric LV hypertrophy. CHDs and other cardiovascular anomalies are more frequent among patients with large NF1 deletion and may cause serious clinical complications. Eccentric LV hypertrophy may occur in NF1 patients without whole gene deletions, but the clinical significance of this finding is uncertain. All patients with clinical suspicion for NF1 should be referred to a cardiologist for evaluation and surveillance.     

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.     

Characterization of the somatic mutational spectrum of the neurofibromatosis type 1 (NF1) gene in neurofibromatosis patients with benign and malignant tumors

One of the main features of neurofibromatosis type 1 (NF1) is benign neurofibromas, 10-20% of which become transformed into malignant peripheral nerve sheath tumors (MPNSTs). The molecular basis of NF1 tumorigenesis is, however, still unclear. Ninety-one tumors from 31 NF1 patients were screened for gross changes in the NF1 gene using microsatellite/restriction fragment length polymorphism (RFLP) markers; loss of heterozygosity (LOH) was found in 17 out of 91 (19%) tumors (including two out of seven MPNSTs). Denaturing high performance liquid chromatography (DHPLC) was then used to screen 43 LOH-negative and 10 LOH-positive tumors for NF1 microlesions at both RNA and DNA levels. Thirteen germline and 12 somatic mutations were identified, of which three germline (IVS7-2A>G, 3731delT, 6117delG) and eight somatic (1888delG, 4374-4375delCC, R2129S, 2088delG, 2341del18, IVS27b-5C>T, 4083insT, Q519P) were novel. A mosaic mutation (R2429X) was also identified in a neurofibroma by DHPLC analysis and cloning/sequencing. The observed somatic and germline mutational spectra were similar in terms of mutation type, relative frequency of occurrence, and putative underlying mechanisms of mutagenesis. Tumors lacking mutations were screened for NF1 gene promoter hypermethylation but none were found. Microsatellite instability (MSI) analysis revealed MSI in five out of 11 MPNSTs as compared to none out of 70 neurofibromas (p=1.8 x 10(-5)). The screening of seven MPNSTs for subtle mutations in the CDKN2A and TP53 genes proved negative, although the screening of 11 MPNSTs detected LOH involving either the TP53 or the CDKN2A gene in a total of four tumors. These findings are consistent with the view that NF1 tumorigenesis is a complex multistep process involving a variety of different types of genetic defect at multiple loci.     

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.     

Somatic mosaicism of a greater than 1.7-Mb deletion of genomic DNA involving the entire NF1 gene as verified by FISH: Further evidence for a contiguous gene syndrome in 17q11.2

We report on a third case with neurofibromatosis type 1 (NF1) due to mosaicism for a gross deletion in 17q11.2 covering the entire NF1 gene. The deletion was suspected in Giemsa banded chromosomes and was confirmed by fluorescence in situ hybridization using the cosmids CO919 from the 5′ region, GO2121 from the central, H10410 from the 3′ region of the NF1 gene, and the 1.7-Mb YAC 947G11 spanning the entire 350-kb genomic DNA of the NF1 gene. The deletion was present in 33% of peripheral blood lymphocytes and 58% of fibroblasts. The clinical manifestations in this 6-year-old male patient were especially severe and extended beyond the typical features of NF1. The patient also displayed facial anomalies, severe and early-onset psychomotor retardation, seizures, spasticity, and microcephaly. These features differ from other large-deletion NF1 patients, even nonmosaic cases. The complex phenotype could be explained by the involvement of coding sequences flanking the NF1 gene, thus supporting the existence of a contiguous gene syndrome in 17q11.2. Am. J. Med. Genet. 87:12–16, 1999. © 1999 Wiley-Liss, Inc.     

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).     

Type 1 neurofibromatosis: A descriptive analysis of the disorder in 1,728 patients

Type 1 Neurofibromatosis, NF1, is a common genetic disorder with variable clinical manifestations. Although NF1 often is only of cosmetic concern, serious and even lethal complications may occur. It is not possible to predict which symptoms will develop in any affected individual. The NNFF International Database is a multicentre collaborative system for collecting information about this condition. At the time of this analysis, complete clinical information was available on 1,479 probands and 249 of their affected relatives with NF1. On average, the age at diagnosis of NF1 was 8 years younger in the probands than in the affected relatives (P<.01). Many of the manifestations of NF1 were more frequent in the probands than in their affected relatives. The age-specific prevalence of most manifestations of NF1 increases with age. Despite biases inherent in a convenience sample from specialist clinics, the frequencies of many of the serious manifestations of NF1 are similar to those of two smaller population-based studies. The frequencies in this study are likely representative of patients seen at specialized clinics. Am. J. Med. Genet. 70:138–143, 1997. © 1997 Wiley-Liss, Inc.     

Deletion of the entire NF1 gene causing distinct manifestations in a family

We identified a father and son with neurofibromatosis type 1 (NF1) due to a deletion of the entire NF1 gene detected by fluorescence in situ hybridization (FISH). As is the case for others reported to have such large deletions, father and son had severe NF1, including a large number of cutaneous neurofibromas, facial anomalies, large hands, feet, and head, and developmental impairment. They were discordant in that seizures and plexiform neurofibromas occurred only in the propositus. Large NF1 deletions can be compatible with familial transmission and appear to be associated with a distinct phenotype. Am. J. Med. Genet. 69:98–101, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of NF1 gene mutations in two sporadic patients with neurofibromatosis type 1CSCD

Objective: To detect mutations of the NF1 gene in two sporadic cases with neurofibromatosis type 1 (NF1) and explore their molecular mechanisms. Methods: Clinical data of the two patients was collected. Genomic DNA was extracted from peripheral blood samples. Specific primers were designed to exclude pseudogenes. PCR was performed to amplify all coding exons of the NF1 gene. PCR products were directly sequenced. Results: Two novel mutations of the NF1 gene (c. 1019-1020delCT in exon 9 and c. 7189G> A in exon 48) were respectively identified in the two patients but not among their unaffected parents or 100 healthy controls. Conclusion: Mutations of the NF1 gene may have predisposed to the NF1 in the two patients. © 2018 MeDitorial Ltd. All rights reserved.     

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.     

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.     

Characterization of the nonallelic homologous recombination hotspot PRS3 associated with type-3 NF1 deletions

Nonallelic homologous recombination (NAHR) is the major mechanism underlying recurrent genomic rearrangements, including the large deletions at 17q11.2 that cause neurofibromatosis type 1 (NF1). Here, we identify a novel NAHR hotspot, responsible for type-3 NF1 deletions that span 1.0 Mb. Breakpoint clustering within this 1-kb hotspot, termed PRS3, was noted in 10 of 11 known type-3 NF1 deletions. PRS3 is located within the LRRC37B pseudogene of the NF1-REPb and NF1-REPc low-copy repeats. In contrast to other previously characterized NAHR hotspots, PRS3 has not developed on a preexisting allelic homologous recombination hotspot. Furthermore, the variation pattern of PRS3 and its flanking regions is unusual since only NF1-REPc (and not NF1-REPb) is characterized by a high single nucleotide polymorphism (SNP) frequency, suggestive of unidirectional sequence transfer via nonallelic homologous gene conversion (NAHGC). By contrast, the previously described intense NAHR hotspots within the CMT1A-REPs, and the PRS1 and PRS2 hotspots underlying type-1 NF1 deletions, experience frequent bidirectional sequence transfer. PRS3 within NF1-REPc was also found to be involved in NAHGC with the LRRC37B gene, the progenitor locus of the LRRC37B-P duplicons, as indicated by the presence of shared SNPs between these loci. PRS3 therefore represents a weak (and probably evolutionarily rather young) NAHR hotspot with unique properties.     

Growth in neurofibromatosis 1 microdeletion patients

Microdeletions of the entire NF1 gene and surrounding genomic region occur in about 5% of patients with neurofibromatosis 1 (NF1). NF1 microdeletion patients usually have more cutaneous and plexiform neurofibromas and a higher risk of developing malignant peripheral nerve sheath tumors than other people with NF1. Somatic overgrowth has also been observed in NF1 microdeletion patients, an observation that is remarkable because most NF1 patients are smaller than average for age and sex. We studied longitudinal measurements of height, weight, and head circumference in 56 patients with NF1 microdeletions and 226 NF1 patients with other kinds of mutations. Although children with NF1 microdeletions were much taller than non‐deletion NF1 patients at all ages after 2 years, the lengths of deletion and nondeletion NF1 patients were similar in early infancy. NF1 microdeletion patients tended to be heavier than other NF1 patients, but height or weight more than 3 standard deviations above the mean for age and sex was infrequent in children with NF1 microdeletions. Head circumference and age of puberty were similar in deletion and non‐deletion NF1 patients. The pattern of growth differs substantially in deletion and non‐deletion NF1 patients, but the pathogenic basis for this difference is unknown.     

Preimplantation genetic diagnosis for neurofibromatosis type 1

PGD is an alternative to prenatal diagnosis that circumvents therapeutic abortion. Diagnosis is carried out on single cells obtained from three-day-old embryos, and only those that are free of the disease under consideration are transferred to the mother. Neurofibromatosis type 1 (NF1) is a common neurocutaneous disorder, inherited as an autosomal dominant trait and caused by mutations in the NF1 gene. For some patients, PGD may be the only acceptable manner to ensure the birth of unaffected children. Because of the large number of known NF1 mutations, the development of mutation-specific single-cell protocols is impractical, labour-intensive and expensive. This paper discusses the development of five PGD protocols, three of which are based on multiplex PCR for microsatellite-markers linked to the NF1 gene. After a linkage study, the diagnosis can be established through the markers, thereby obviating the need to detect the mutation itself. This not only ensures the accurate diagnosis of the embryos, but also a prompt acceptance of PGD referrals since one protocol can be useful for several couples. In addition, two mutation-specific PCRs were developed for two couples where a marker-based protocol was not applicable. In total, 16 PGD cycles were carried out for six couples, which resulted in one ongoing pregnancy and the delivery of a healthy unaffected boy.     

Ten novel mutations in the human neurofibromatosis type 1 (NF1) gene in Italian patients

The entire NF1 coding region was analyzed for mutations in a panel of 108 unrelated Italian NF1 patients. Using PTT, SSCP, and DNA sequencing, we found 10 mutations which have never been reported before. Clinical diagnosis of NF1 was established according to the NIH consensus criteria in 100 individuals, while 8 were young children with only multiple cafè-au-lait spots. We detected 46 truncated fragments, and 24 of them were fully characterized by SSCP and direct sequencing. Of the 24, 14 were known mutations (R304X, R681X, Q682X, R1306X, R1362X, R1513X, R1748X, Q1794X, R1947X, Y2264X, R2237X, 2674delA, 6789delTTAC, 2027insC). The other 10 mutations represent novel changes that contribute to the germline mutational spectrum of the NF1 gene (K810X, Q2595X, 6772delT, 7190delCT, 7331delA, 1021insTT, 3921insT, 4106insTA, 7149insC, 2033insCG / 2034delA). PTT in a large number of Italian NF1 patients supports the usefulness of this method for characterization of mutations in disorders where the responsible gene is very large and the disease-causing mutations often create a stop codon. In agreement with previous reports, no mutational hotspots within the NF1 gene were detected.     

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.     

Tissue-specific differences in the proportion of mosaic large NF1 deletions are suggestive of a selective growth advantage of hematopoietic del(+/-) stem cells

Type-2 NF1 deletions spanning 1.2 Mb are frequently of postzygotic origin and hence tend to be associated with mosaicism for normal cells and those harboring the deletion (del(+/-) cells). Eleven patients with mosaic type-2 deletions were investigated by FISH and high proportions (94-99%) of del(+/-) cells were detected both in whole blood and in isolated CD3+, CD14+, CD15+, and CD19+ leukocytes. Significantly lower proportions of del(+/-) cells (24-82%) were however noted in urine-derived epithelial cells. A patient harboring an atypical large NF1 deletion with nonrecurrent breakpoints was also found to have a much higher proportion of del(+/-) cells in blood (96%) than in urine (51%). The tissue-specific differences in the proportions of del(+/-) cells as well as the X chromosome inactivation (XCI) patterns observed in these mosaic patients suggest that the majority of the deletions had occurred before or during the preimplantation blastocyst stage before the onset of XCI. We postulate that hematopoietic del(+/-) stem cells present at an early developmental stage are characterized by a selective growth advantage over normal cells lacking the deletion, leading to a high proportion of del(+/-) cells in peripheral blood from the affected patients.