NF1 gene analysis based on DHPLC

Luc, Alessandro De; Buccino, Anna; Gianni, Debora; Mangino, Massimo; Giustini, Sandra; Richetta, Antonio Giovanni; Divona, Luigina; Calvieri, Stefano; Mingarelli, Rita; Dallapiccola, Bruno

doi:10.1002/humu.9111

Cited by 36 publications

(5 citation statements)

References 12 publications

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“…14 Starting from 2005, the negative samples at the sequencing analysis have been subjected to Multiplex Ligation-dependent Probe Amplification (MLPA, MRC Holland) for the detection of deletions or duplications spanning one or more exons. The combined approach of NGS and MLPA has a reported detection rate of 88%, 23 higher than the 72% of the previously used DHPLC technique, 24 reflecting the technical changes over the years but not substantially affecting the results of the study, which is based on the mutation-positive cases.…”

Section: Nf1 Genetic Testmentioning

confidence: 89%

Recurrent NF1 gene variants and their genotype/phenotype correlations in patients with Neurofibromatosis type I

Riva

Martorana

Uliana

et al. 2021

Genes Chromosomes & Cancer

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Neurofibromatosis type I, a genetic condition due to pathogenic variants in the NF1 gene, is burdened by a high rate of complications, including neoplasms, which increase morbidity and mortality for the disease. We retrospectively re-evaluated the NF1 gene variants found in the period 2000-2019 and we studied for genotype/ phenotype correlations of disease complications and neoplasms 34 variants, which were shared by at least two unrelated families (range 2-11) for a total 141 of probands and 21 relatives affected by Neurofibromatosis type I. Recurrent variants could be ascribed to the most common mutational mechanisms (C to T transition, microsatellite slippage, non-homologous recombination). In genotype/phenotype correlations, the variants p.Arg440*, p.Tyr489Cys, and p.Arg1947*, together with the gross gene deletions, displayed the highest rates of complications. When considering neoplasms, carriers of variants falling in the extradomain region at the 5 0 end of NF1 had a lower age-related cancer frequency than the rest of the gene sequence, showing a borderline significance (p = 0.045), which was not conserved after correction with covariates. We conclude that (1) hotspots in NF1 occur via different mutational mechanisms, (2) several variants are associated with high rates of complications and cancers, and (3) there is an initial evidence toward a lower cancer risk for carriers of variants in the 5 0 end of the NF1 gene although not significant at the multivariate analysis.

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Section: Nf1 Genetic Testmentioning

confidence: 89%

Recurrent NF1 gene variants and their genotype/phenotype correlations in patients with Neurofibromatosis type I

Riva

Martorana

Uliana

et al. 2021

Genes Chromosomes & Cancer

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“…NF1 cDNA was amplified in 22 overlapping fragments according to the conditions described in Valero et al, 2011 [ 5 ]. The cDNA alterations were confirmed at the DNA level using genomic primers designed according to several reports [ 4 , 7 , 8 , 9 ]. Both cDNA and gDNA PCR products were gel-purified using the GeneClean Kit (Qiagen) and sequenced with an ABI PRISM BigDye terminator sequencing kit v1.1 (Life Technologies) on an ABI 310 Genetic Analyser (Life Technologies).…”

Section: Methodsmentioning

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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“…From April 2000 to June 2016 (397 out of the total 502 patients, 79.1%), genetic analyses on genomic DNA were conducted using denaturing high pressure liquid chromatography (DHPLC), which exploits the differential retention of homoduplex and heteroduplex DNA under conditions of partial thermal denaturation (reported detection rate for NF1 mutations: 72% [ 30 ]). Primer pairs were designed according to the published reference genomic NF1 sequence (GenBank accession number NG_009018.1).…”

Section: Methodsmentioning

confidence: 99%

Patterns of Novel Alleles and Genotype/Phenotype Correlations Resulting from the Analysis of 108 Previously Undetected Mutations in Patients Affected by Neurofibromatosis Type I

Bonatti

Adorni

Matichecchia

et al. 2017

IJMS

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Neurofibromatosis type I, a genetic disorder due to mutations in the NF1 gene, is characterized by a high mutation rate (about 50% of the cases are de novo) but, with the exception of whole gene deletions associated with a more severe phenotype, no specific hotspots and few solid genotype/phenotype correlations. After retrospectively re-evaluating all NF1 gene variants found in the diagnostic activity, we studied 108 patients affected by neurofibromatosis type I who harbored mutations that had not been previously reported in the international databases, with the aim of analyzing their type and distribution along the gene and of correlating them with the phenotypic features of the affected patients. Out of the 108 previously unreported variants, 14 were inherited by one of the affected parents and 94 were de novo. Twenty-nine (26.9%) mutations were of uncertain significance, whereas 79 (73.2%) were predicted as pathogenic or probably pathogenic. No differential distribution in the exons or in the protein domains was observed and no statistically significant genotype/phenotype correlation was found, confirming previous evidences.

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NF1 gene analysis based on DHPLC

Cited by 36 publications

References 12 publications

Recurrent NF1 gene variants and their genotype/phenotype correlations in patients with Neurofibromatosis type I

Recurrent NF1 gene variants and their genotype/phenotype correlations in patients with Neurofibromatosis type I

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

Patterns of Novel Alleles and Genotype/Phenotype Correlations Resulting from the Analysis of 108 Previously Undetected Mutations in Patients Affected by Neurofibromatosis Type I

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