Bianchessi Donatella, Morosini Sara, Saletti Veronica, Ibba Maria Cristina, Natacci Federica, Esposito Silvia, Cesaretti Claudia, Riva Daria, Finocchiaro Gaetano, Eoli Marica
Molecular Neuro-oncology IRCCS Foundation, "C. Besta" Neurological Institute Milan Italy.
Developmental Neurology IRCCS Foundation, "C. Besta" Neurological Institute Milan Italy.
Mol Genet Genomic Med. 2015 Jul 7;3(6):513-25. doi: 10.1002/mgg3.161. eCollection 2015 Nov.
Genetic analysis of Neurofibromatosis type 1 (NF1) may facilitate the identification of patients in early phases of the disease. Here, we present an overview of our diagnostic research spanning the last 11 years, with a focus on the description of 225 NF1 mutations, 126 of which are novel, found in a series of 607 patients (513 unrelated) in Italy. Between 2003 and 2013, 443 unrelated patients were profiled by denaturing high pressure liquid chromatography (DHPLC) analysis of 60 amplicons derived from genomic NF1 DNA and subsequent sequencing of heterozygotic PCR products. In addition, a subset of patients was studied by multiplex ligation-dependent probe amplification (MLPA) to identify any duplications, large deletions or microdeletions present at the locus. Over the last year, 70 unrelated patients were investigated by MLPA and sequencing of 22 amplicons spanning the entire NF1 cDNA. Mutations were found in 70% of the 293 patients studied by DHPLC, thereby fulfilling the NIH criterion for the clinical diagnosis of NF1 (detection rate: 70%); furthermore, 87% of the patients studied by RNA sequencing were genetically characterized. Mutations were also found in 36 of the 159 patients not fulfilling the NIH clinical criteria. We confirmed a higher incidence of intellectual disability in patients harboring microdeletion type 1 and observed a correlation between a mild phenotype and the small deletion c.2970_2972delAAT or the missense alteration in amino acid residue 1809 (p.Arg1809Cys). These data support the use of RNA-based methods for genetic analysis and provide novel information for improving the management of symptoms in oligosymptomatic patients.
1型神经纤维瘤病（NF1）的基因分析可能有助于识别疾病早期阶段的患者。在此，我们概述了过去11年的诊断研究，重点描述了在意大利607例患者（513例无亲缘关系）中发现的225个NF1突变，其中126个是新发现的。2003年至2013年期间，通过对源自基因组NF1 DNA的60个扩增子进行变性高效液相色谱（DHPLC）分析以及随后对杂合PCR产物进行测序，对443例无亲缘关系的患者进行了分析。此外，对一部分患者进行了多重连接依赖探针扩增（MLPA）研究，以识别该基因座处存在的任何重复、大片段缺失或微缺失。在过去一年中，通过MLPA和对跨越整个NF1 cDNA的22个扩增子进行测序，对70例无亲缘关系的患者进行了研究。在通过DHPLC研究的293例患者中，70%发现了突变，从而满足了美国国立卫生研究院（NIH）对NF1临床诊断的标准（检测率：70%）；此外，通过RNA测序研究的患者中，87%获得了基因特征。在未满足NIH临床标准的159例患者中，也有36例发现了突变。我们证实了携带1型微缺失的患者中智力残疾的发生率较高，并观察到轻度表型与小缺失c.2970_2972delAAT或氨基酸残基1809处的错义改变（p.Arg1809Cys）之间存在相关性。这些数据支持使用基于RNA的方法进行基因分析，并为改善症状轻微患者的症状管理提供了新信息。
