Large Neurofibromatosis Type 1 Research Articles

P-562 Molecular aspects of preimplantation genetic testing (PGT) for Neurofibromatosis type 1 (NF1) in three large European PGT centers: challenges and pitfalls

Abstract Study question What are the technical challenges and pitfalls of PGT for NF1 (n = 296 families) as experienced by three large European PGT centers? Summary answer The high (de novo) mutation rate and presence of germline mosaicism, especially in case of deletions, is challenging in PGT test development for NF1. What is known already NF1 is a neurocutaneous disorder with complete penetrance and extreme clinical variability. It is one of the most frequently requested autosomal dominant indications for PGT. The NF1 gene is large and has a high mutation rate. A high number of variants occur de novo, are mostly unique and more frequently associated with germline mosaicism in the index. These characteristics can pose difficulties in counseling, test development and PGT treatment. Large NF1 deletions are associated with a more severe phenotype and relative frequent mosaicism, but this has not been described in literature for intragenic deletions. Study design, size, duration Retrospective analysis of data on all test development procedures with the indication NF1 completed by August 2022 (n = 296). The data on test development were collected from medical files by the laboratories of the three participating PGT centers. The study was approved by the Medical and Ethical Review Committee of all three centers. The Medical Research Involving Human Subjects Act was not applicable. Participants/materials, setting, methods The three participating PGT centers were Maastricht University Medical Center (the Netherlands), University Hospital Brussels (Belgium) and Strasbourg University Hospital (France). Techniques applied were locus specific PCR-STR with or without the specific causative variant or genome wide haplotyping methods (NGS based OnePGT or karyomapping) or a combination of the above. Main results and the role of chance Test development was completed for 296 cases involving 228 different variants in the NF1 gene (125 truncating, 31 missense, 47 splice and 7 in-frame or synonymous variants, 8 larger deletions, 1 translocation, 9 awaiting further classification). We observed 36 recurring variants for 104 couples, of which 55 were sporadic cases, The majority of the 296 cases were sporadic (60%), signs of mosaicism were observed in 14 of these cases (8%). In our cohort, 6 sporadic cases involved a complete gene deletion (n = 1) or a deletion of ≥ 1 exons in the NF1 gene (n = 5). Mosaicism was detected in 4 of these cases (67%). In one case, the mutation was not detected in sperm cells during test set-up. PGT treatment was started for 10 couples with signs of mosaicism, in 6 no affected embryos were detected in 1-2 cycles and 1 resulted in a misdiagnosis because of phasing based on unaffected offspring. Two cases had NF1 caused by a different variant in the NF1 gene than their affected family members. A successful PGT test was developed in 289 cases (98%), 5 couples opted out during test development and in only 2 cases, development of a test was not possible for technical reasons. Limitations, reasons for caution The observed challenges and pitfalls depend on the techniques used and local procedures, so results may not be applicable to all PGT laboratories. However, due to the large sample size with exhaustive data and the fact that ESHRE guidelines were followed, there is probably little reason for caution. Wider implications of the findings Our findings could improve broader reproductive counseling of NF1 patients. Also, this large cohort contains previously unpublished variants in the NF1 gene, which is relevant for genetic diagnosis in NF1 patients in general. Our findings could possibly aid in test development for other autosomal dominant PGT indications with similar characteristics. Trial registration number Not applicable.

A Review of Spinal Lesions in Neurofibromatosis Type 1 in a Large Neurofibromatosis Type 1 Center

Spinal lesions are a known manifestation of neurofibromatosis type 1 (NF1). The aim of this retrospective review was to analyze and report the prevalence of spinal lesions on imaging in a large NF1 center. The data were collected from a period of 62 months from a cohort of 514 patients. Data were collected from multidisciplinary team meeting reports that included radiologic reports of each patient investigating 20 distinct variables. The prevalence of each of these lesions was calculated, and any statistically significant associations were investigated using the χ2 test. Four-hundred forty-seven patients had classic NF1, and 67 patients had spinal NF1. Many of the patients had spinal abnormalities; 25.7% of these patients were found to have dural ectasia, whereas 44.9% of patients had a spinal deformity. A statistically significant association between dural ectasia and spinal neurofibromatosis was established (P < 0.05). An additional statically significant association was established between dural ectasia and spinal deformity (P < 0.00001). The patients with spinal nerve root tumors were identified, and it was found that 49.8% of patients possessed these tumors, whereas 56.3% of these tumors were intraspinal tumors. The most common region affected was the cervical spine, and the most common spinal level was C2. This high prevalence of spinal tumours in mobile areas of the spine is possibly the result of a combination of genetic predisposition and repeated microtraumas resulting in tumor formation. This is the largest reported study of spinal lesions in NF1 based on imaging and offers insights into the etiology and relationships between lesions.

Atypical NF1 Microdeletions: Challenges and Opportunities for Genotype/Phenotype Correlations in Patients with Large NF1 Deletions.

Patients with neurofibromatosis type 1 (NF1) and type 1 NF1 deletions often exhibit more severe clinical manifestations than patients with intragenic NF1 gene mutations, including facial dysmorphic features, overgrowth, severe global developmental delay, severe autistic symptoms and considerably reduced cognitive abilities, all of which are detectable from a very young age. Type 1 NF1 deletions encompass 1.4 Mb and are associated with the loss of 14 protein-coding genes, including NF1 and SUZ12. Atypical NF1 deletions, which do not encompass all 14 protein-coding genes located within the type 1 NF1 deletion region, have the potential to contribute to the delineation of the genotype/phenotype relationship in patients with NF1 microdeletions. Here, we review all atypical NF1 deletions reported to date as well as the clinical phenotype observed in the patients concerned. We compare these findings with those of a newly identified atypical NF1 deletion of 698 kb which, in addition to the NF1 gene, includes five genes located centromeric to NF1. The atypical NF1 deletion in this patient does not include the SUZ12 gene but does encompass CRLF3. Comparative analysis of such atypical NF1 deletions suggests that SUZ12 hemizygosity is likely to contribute significantly to the reduced cognitive abilities, severe global developmental delay and facial dysmorphisms observed in patients with type 1 NF1 deletions.

Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis.

SUMMARYNeurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.

Severe Phenotype in Patients with Large Deletions of NF1.

Simple SummaryNeurofibromatosis type 1 (NF1) is a genetic disorder caused by pathogenic variants in the NF1 tumor suppressor gene. In 5–10% of NF1 patients, a large heterozygous deletion of the whole NF1 gene is identified, leading to the commonly called “NF1 microdeletion syndrome”. NF1-deleted patients were previously reported to develop a particularly severe form of the disease with frequent cognitive impairment and an increased risk of benign and malignant tumors. Here, we performed a comprehensive clinical assessment of the largest NF1-deleted cohort to date, including 126 NF1 patients with a deletion of the NF1 gene. This work provides new insights into the specific phenotype associated with NF1 deletions and may contribute to improve the follow-up care of NF1 patients. Complete deletion of the NF1 gene is identified in 5–10% of patients with neurofibromatosis type 1 (NF1). Several studies have previously described particularly severe forms of the disease in NF1 patients with deletion of the NF1 locus, but comprehensive descriptions of large cohorts are still missing to fully characterize this contiguous gene syndrome. NF1-deleted patients were enrolled and phenotypically characterized with a standardized questionnaire between 2005 and 2020 from a large French NF1 cohort. Statistical analyses for main NF1-associated symptoms were performed versus an NF1 reference population. A deletion of the NF1 gene was detected in 4% (139/3479) of molecularly confirmed NF1 index cases. The median age of the group at clinical investigations was 21 years old. A comprehensive clinical assessment showed that 93% (116/126) of NF1-deleted patients fulfilled the NIH criteria for NF1. More than half had café-au-lait spots, skinfold freckling, Lisch nodules, neurofibromas, neurological abnormalities, and cognitive impairment or learning disabilities. Comparison with previously described “classic” NF1 cohorts showed a significantly higher proportion of symptomatic spinal neurofibromas, dysmorphism, learning disabilities, malignancies, and skeletal and cardiovascular abnormalities in the NF1-deleted group. We described the largest NF1-deleted cohort to date and clarified the more severe phenotype observed in these patients.

Pronounced maternal parent-of-origin bias for type-1 NF1 microdeletions.

Neurofibromatosis type 1 (NF1) is caused, in 4.7-11% of cases, by large deletions encompassing the NF1 gene and its flanking regions within 17q11.2. Different types of large NF1 deletion occur which are distinguishable by their breakpoint location and underlying mutational mechanism. Most common are the type-1 NF1 deletions of 1.4 Mb which exhibit recurrent breakpoints caused by nonallelic homologous recombination (NAHR), also termed unequal crossover. Here, we analyzed 37 unrelated families of patients with de novo type-1 NF1 deletions by means of short tandem repeat (STR) profiling to determine the parental origin of the deletions. We observed that 33 of the 37 type-1 deletions were of maternal origin (89.2% of cases; p < 0.0001). Analysis of the patients' siblings indicated that, in 14 informative cases, ten (71.4%) deletions resulted from interchromosomal unequal crossover during meiosis I. Our findings indicate a strong maternal parent-of-origin bias for type-1 NF1 deletions. A similarly pronounced maternal transmission bias has been reported for recurrent copy number variants (CNVs) within 16p11.2 associated with autism, but not so far for any other NAHR-mediated pathogenic CNVs. Region-specific genomic features are likely to be responsible for the maternal bias in the origin of both the 16p11.2 CNVs and type-1 NF1 deletions.

Clinical findings and mutation analysis of NF1 patients in Turkey

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that is caused by mutations of the NF1 gene. NF1 is clinically characterized by neurofibromas, pigmentation anomalies, and an increased risk of malignant tumors. The mutation rate of NF1 is one of the highest known for human disorders: approximately 50% of all affected individuals are sporadic cases and carry de novo mutations Therefore mutation analysis of NF1 may be an important tool in early diagnosis and genetic counseling. This is the first large NF1 study performed in Turkey. The data collected in this study enabled us to overview the genetic and clinical aspects of NF1 molecular diagnostics. The patients, who were clinically diagnosed for NF1, were included in this study. These patients were clinically evaluated, and subgroup of them genotyped or DNA sequenced for mutations in NF1, either to confirm the clinical diagnosis or to identify pathogenic mutations. The mutation detection rate was 52%, based on analysis of only genomic DNA. We observed that frameshift mutations were the largest proportion of the identified mutations (38.5%). The frequency of microdeletions was 26.9% and the splice site and nonsense mutations were 11.5% in this cohort. Turkish NF1 patients have similar NF1 germline mutations compared to other populations. Considering that some of these detected mutations belonged to the patients who did not fulfill the NIH criteria for NF1 diagnosis, mutation analysis of NF1 is an important tool in early diagnosis and genetic counseling.

Neurofibromatosis Type 1 Molecular Diagnosis: The RNA Point of View

Neurofibromatosis Type 1 Molecular Diagnosis: The RNA Point of View

Identification of large NF1 duplications reciprocal to NAHR-mediated type-1 NF1 deletions.

Approximately 5% of all patients with neurofibromatosis type-1 (NF1) exhibit large deletions of the NF1 gene region. To date, only nine unrelated cases of large NF1 duplications have been reported, with none of the affected patients exhibiting multiple café au lait spots (CALS), Lisch nodules, freckling, or neurofibromas, the hallmark signs of NF1. Here, we have characterized two novel NF1 duplications, one sporadic and one familial. Both index patients with NF1 duplications exhibited learning disabilities and atypical CALS. Additionally, patient R609021 had Lisch nodules, whereas patient R653070 exhibited two inguinal freckles. The mother and sister of patient R609021 also harbored the NF1 duplication and exhibited cognitive dysfunction but no CALS. The breakpoints of the nine NF1 duplications reported previously have not been identified and hence their underlying generative mechanisms have remained unclear. In this study, we performed high-resolution breakpoint analysis that indicated that the two duplications studied were mediated by nonallelic homologous recombination (NAHR) and that the duplication breakpoints were located within the NAHR hotspot paralogous recombination site 2 (PRS2), which also harbors the type-1 NF1 deletion breakpoints. Hence, our study indicates for the first time that NF1 duplications are reciprocal to type-1 NF1 deletions and originate from the same NAHR events.

Internal tumor burden in neurofibromatosis Type I patients with large NF1 deletions

Neurofibromatosis Type 1 (NF1) is a frequent tumor suppressor gene disorder characterized by multiple benign tumors and high risk of malignancy. Internal tumor burden is a major disease-associated manifestation and can be most adequately assessed by magnetic resonance imaging of the whole body. Approximately 5% of NF1 patients have constitutional large NF1-deletions that are generally associated with more severe clinical manifestations. Here, we investigated whether these deletion patients also have more and/or larger internal tumors by assessing internal tumors and their total volume (exclusive of cutaneous and subcutaneous) in 38 NF1 deletion patients (including eight mosaic cases) and 114 age- and gender-matched NF1 patients without deletions. The incidence of internal tumors was significantly lower in mosaic deletion patients (1/8 = 13%) but did not differ between the 30 nonmosaic deletion patients and the 90 age- and gender-matched NF1 patients without large deletions used as controls. Neither the number nor the total volume of tumors per patient differed significantly between the latter two groups. However, extremely high tumor burden (>3,000 ml) was significantly more frequent among nonmosaic NF1 deletion patients than among NF1 patients without large deletions (13% vs. 1%, P = 0.014). Thus, as a group, patients with NF1 deletions do not exhibit a significantly higher internal tumor burden than NF1 patients without such deletions. However, deletion patients can frequently have extremely large internal tumors and thus demand special attention.

Clinical characterisation of 29 neurofibromatosis type-1 patients with molecularly ascertained 1.4 Mb type-1 NF1 deletions

BackgroundLarge deletions of the NF1 gene region occur in ∼5% of patients with neurofibromatosis type-1 (NF1) and are associated with particularly severe manifestations of the disease. However, until now, the...

Orbital optic nerve gliomas in children with neurofibromatosis type 1

To describe the clinical course and treatment of symptomatic orbital optic nerve gliomas in children with neurofibromatosis type-1 (NF-1). A retrospective review of the records of patients with NF-1 and symptomatic orbital optic nerve gliomas seen in a large multidisciplinary NF-1 clinic of a tertiary care children's hospital. The main outcome measures included presenting symptoms and signs, ophthalmologic examination at diagnosis, the presence of progressive disease following diagnosis, type of therapy, and the reasons therapy was instituted. Twelve patients with symptomatic orbital optic nerve gliomas, all of which led to proptosis (eight girls, four boys), were identified. The mean age of diagnosis of NF-1 was 20 months; the mean age of diagnosis of the orbital optic nerve glioma was 26 months. At the time of diagnosis of the tumor, 10 of 12 patients (83%) had decreased visual acuity in the affected eye. Three patients underwent optic nerve resection; eight received chemotherapy, and one was observed without therapy. Of the eight children who received chemotherapy, progressive disease prior to treatment could be documented in only three; none of these eight children had a reproducible improvement in vision following chemotherapy. There was no demonstrable improvement in vision in any treated patient with NF-1-associated orbital optic nerve gliomas. Although not definitively proven, our data and previous studies suggest that NF-1-associated orbital optic nerve gliomas should not be treated unless there is clear evidence of either ophthalmologic or radiographic progression. Surgical excision of tumors which have led to proptotic eyes without functional vision should be reserved for cosmetic purposes or to treat complications of exposed globes.

A large deletion (1.5 Mb) encompassing the neurofibromatosis type 1 (NF1) gene in a patient with sporadic NF1 associated with dysmorphism, mental retardation, and unusual ocular and skeletal features

A 20 year old male patient with sporadic neurofibromatosis type 1 (NF1) is described with a large deletion (1.5 Mb) involving theNF1 gene, dysmorphism, mental retardation, and unusual ocular and skeletal features. Several NF1 patients with a large NF1 deletion and associated dysmorphism, and a large number of neurofibromas for their age have been described. This study indicates that such large deletions can also involve flanking loci which affect ocular and skeletal development.Clin Dysmorphol12:199-201 © 2003 Lippincott Williams & Wilkins.

Optic pathway gliomas in neurofibromatosis type 1: the effect of presenting symptoms on outcome.

Children with neurofibromatosis type 1 (NF1) may present with optic pathway gliomas (OPG) that can progress to visual loss or other neurologic symptoms. These tumors may become evident either as a result of patient signs or symptoms or as an incidental finding on "baseline" neuroimaging studies. In an attempt to determine if there were differences between symptomatic and asymptomatic children with OPG, a retrospective cohort study of ninety children with NF1 and OPG was performed using data from two large NF1 referral centers. Age at diagnosis, presenting symptoms, tumor location, associated features, and clinical response were assessed for children who were initially symptomatic from their OPG (n = 51) and compared to similar data of asymptomatic children whose tumors were incidentally discovered by MRI (n = 39). There were no differences in age at presentation, tumor location, NF1-associated symptoms, or clinical response between the groups. Initially symptomatic children were much more likely to require treatment (OR: 14.8, 95% CI [1.9-116.7]) than those with incidentally discovered, asymptomatic OPG. Although 36% of OPG were diagnosed in children over the age of 6 years, none received prior neuroimaging and only two children had previously normal eye examinations, suggesting that the vast majority of OPG in this group were longstanding, undiagnosed tumors. Based on these findings, we do not advocate "baseline" MRI in children with NF1, but strongly recommend that all children of the age 10 years and younger with NF1 have complete annual ophthalmologic evaluations.

Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by a marked variability in expression. A more severe phenotype is frequently observed in the group of patients carrying a large NF1 deletion. To study the extent of the microdeletion in these NF1 patients, we generated a partial physical map of the NF1 flanking region. We describe seven PACs and three new polymorphic dinucleotide repeats located outside the NF1 gene and analyzed 20 unrelated individuals with an NF1 microdeletion in a collaborative study. We detected one individual with a substantially smaller deletion including only the NF1 gene and its three embedded genes. In the other 19 patients, the deletion extended at least 1 Mb. The parental origin of the deletion was determined in 15 individuals and was maternal in 13 and paternal in two cases. The new molecular tools described here can be used to unequivocally diagnose a possible extragenic extension of an NF1 deletion.

Constitutional and mosaic large NF1 gene deletions in neurofibromatosis type 1.

A set of neurofibromatosis type 1 (NF1) patients was screened for large NF1 gene deletions by comparing patient and parent genotypes at 10 intragenic polymorphic loci. Of 67 patient/parent sets...

Deletion of the entireNF1 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.

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.

NF1 mutation analysis using a combined heteroduplex/SSCP approach.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized predominantly by neurofibromas, café-au-lait spots, and Lisch nodules. The disease is caused by disruptive mutations of the large NF1 gene, with half of cases caused by new mutation. Less than 100 constitutional mutations have thus far been published, ranging from very large deletions to point mutations. We have pursued NF1 mutation analysis by heteroduplex analysis (HDA) and single-strand conformational polymorphism analysis (SSCP) of individual exons. We streamlined these techniques to eliminate the use of radioactivity, to apply both methods to the same PCR product, and to multiplex samples in gels. Applied simultaneously to a set of 67 unrelated NF1 patients, HDA and SSCP have thus far identified 26 mutations and/or variants in 45 of the 59 exons tested. Disease-causing mutations were found in 19% (13/67) of cases studied. Both techniques detected a variety of mutations including splice mutations, insertions, deletions, and point changes, with some overlap in the ability of each method to detect variants.