Development of a practical NF1 genetic testing method through the pilot analysis of five Japanese families with neurofibromatosis type 1

Okumura, Akiko; Ozaki, Mamoru; Niida, Yo

doi:10.1016/j.braindev.2014.11.002

Cited by 9 publications

(17 citation statements)

References 28 publications

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“…The identification of predisposing mutations in the NF1 gene is complicated by its large size (257 Kb) encompassing 57 constitutive exons and one major alternatively spliced exon. Further challenges to molecular diagnosis are represented by the existence of 15 pseudogenes [ 4 ], no mutational hot spots, and a high mutation rate. Therefore, molecular testing of the NF1 gene is cumbersome, time-consuming and expensive.…”

Section: Introductionmentioning

confidence: 99%

Accurate Classification of NF1 Gene Variants in 84 Italian Patients with Neurofibromatosis Type 1

Stella

Lastella²,

Loconte

et al. 2018

Genes

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Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant genetic diseases. It is caused by mutations in the NF1 gene encoding for the large protein, neurofibromin. Genetic testing of NF1 is cumbersome because 50% of cases are sporadic, and there are no mutation hot spots. In addition, the most recognizable NF1 clinical features—café-au-lait (CALs) spots and axillary and/or inguinal freckling—appear early in childhood but are rather non-specific. Thus, the identification of causative variants is extremely important for early diagnosis, especially in paediatric patients. Here, we aimed to identify the underlying genetic defects in 72 index patients referred to our centre for NF1. Causative mutations were identified in 58 subjects, with 29 being novel changes. We evaluated missense and non-canonical splicing mutations with both protein and splicing prediction algorithms. The ratio of splicing mutations detected was higher than that reported in recent patients’ series and in the Human Gene Mutation Database (HGMD). After applying in silico predictive tools to 41 previously reported missense variants, we demonstrated that 46.3% of these putatively missense mutations were forecasted to alter splicing instead. Our data suggest that mutations affecting splicing can be frequently underscored if not analysed in depth. We confirm that hamartomas can be useful for diagnosing NF1 in children. Lisch nodules and cutaneous neurofibromas were more frequent in patients with frameshifting mutations. In conclusion, we demonstrated that comprehensive in silico analysis can be a highly specific method for predicting the nature of NF1 mutations and may help in assuring proper patient care.

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Section: Introductionmentioning

confidence: 99%

Accurate Classification of NF1 Gene Variants in 84 Italian Patients with Neurofibromatosis Type 1

Stella

Lastella²,

Loconte

et al. 2018

Genes

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“…So it is economical as compared to the previous price of DNA sequencing. Recently, Okumura et al 22 have reported a practicable and inexpensive NF1 mutation screening system based on CEL endonuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining recently. Despite all that the present study could not adopt this technique because it was a new protocol that it had not been widely used in China.…”

Section: Discussionmentioning

confidence: 99%

Clinical and Molecular Characterization of NF1 Patients

et al. 2016

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Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, and the presence of pseudogenes.Our goal was to establish a sensitive, feasible, and comparatively economical protocol to detect NF1 mutations using blood samples.We developed a method to screen patients for mutations. Thirty-two NF1 patients from 32 unrelated families and 120 unrelated population-match controls were investigated in this study. Specific primers were designed for NF1 to avoid pseudogenes. NF1 mutations were detected by sequencing at the deoxyribonucleic acid (DNA) and complementary DNA (cDNA) levels, and multiplex ligation-dependent probe amplification (MLPA) and familial segregation analyses were used.Forty-four specific primers designed according to the NF1 structure were successfully used for polymerase chain reaction (PCR) and DNA sequencing, which was more feasible and useful than cDNA sequencing. Thirty distinct NF1 mutations were identified in 32 patients. Thirteen mutations were novel and most were frameshift mutations (33.3%). Mutations were detected at a rate of 93.8%.Our study suggests that this sensitive, feasible, and comparatively economical protocol is effective for the detection of NF1 mutations.

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“…Exon 1 of NF1 is located in a GC rich region and required separate amplification using with KOD -Plus-Ver 2. polymerase (Toyobo). The PCR product was determined by direct sequencing without CHIPS screening [3].…”

Section: Protocolmentioning

confidence: 99%

“…PCR reaction were performed in a 10 μl volume with 50 ng/μl of template DNA, 0.4 μM of both forward and reverse primers, and 0.2 unit of Taq DNA polymerase (Roche) for Ta=58ºC and 0.25 units of Blend Taq -plus-(Toyobo) for Ta=60ºC. PCR primer sequences are available on elsewhere [3].…”

Section: Protocolmentioning

confidence: 99%

“…NF1 is characterized by multiple café-au-lait spots, Lisch nodules in the iris, and fibromatous tumors of the skin [1]. A summary of the previous studies shows more than 90% of these patients have NF1 small sequence variants thoroughout the NF1 gene [2].To address this problem, we recently developed CHIPS (CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining) technology for a mutation screening of a large sized gene, and it was applied to NF1 analysis [3]. CHIPS is based on enzyme mismatch cleavage (EMC) method, and finely optimized at every step to achieve maximum sensitivity and simplicity.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Development of a practical NF1 genetic testing method through the pilot analysis of five Japanese families with neurofibromatosis type 1

Cited by 9 publications

References 28 publications

Accurate Classification of NF1 Gene Variants in 84 Italian Patients with Neurofibromatosis Type 1

Accurate Classification of NF1 Gene Variants in 84 Italian Patients with Neurofibromatosis Type 1

Clinical and Molecular Characterization of NF1 Patients

CHIPS for a Mutation Screening of a Large Sized Gene: An example of NF1 Analysis

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