Identification of forty-five novel and twenty-three knownNF1 mutations in Chinese patients with neurofibromatosis type 1

Abstract

Neurofibromatosis type 1 (NF1), characterized by skin neurofibromas and an excess of café-au-lait spots, is due to mutations in the neurofibromin (NF1) gene. Identifying the genetic defect in individuals with the disease represents a significant challenge because the gene is extremely large with a high incidence of sporadic mutations across the entire gene ranging from single nucleotide substitutes to large deletions. In the present study, we have used a combination of techniques (heteroduplex analysis, sequencing, loss of heterozygosity and quantification of gene dosage) to define the genetic defect in 68 individuals from a cohort of 107 NF1 Taiwanese patients of Chinese origin. Fifty-eight were initially identified using heteroduplex analytical techniques and confirmed by sequence analysis. A further five were identified by direct sequence analysis alone. The reminders were shown to carry large deletions in the NF1 gene by demonstrating loss of heterozygosity that was confirmed by gene dosage measurements using quantitative-PCR techniques. Mis-sense, non-sense, frameshift or splice-site mutations were identified across the entire gene of which the majority (45/68) were novel in nature. The detection rate with the various analytical techniques and the types of mutation detected are consistent with published data involving both individuals and large cohort studies from other ethnic backgrounds. INTRODUCTIONWith a frequency of 1/3000 live births, Neurofibromatosis type 1 (NF1; MIM# 162200) is one of the most common autosomal dominant disorders involving the human nervous system [Friedman, 1999]. The clinical features of the disease include: café-au-lait spots, cutaneous or subcutaneous neurofibromas, large plexiform neurofibromas, Lisch nodules and skin freckling. Other features such as scoliosis, macrocephaly, pseudoarthrosis, short stature, mental sub-normality and malignancies are present in a minority of patients 1997]. Except for the discoloration of the skin (Café-au-lait macules), which can be identified at a very young age, most of the other clinical features usually occur insidiously after puberty.The NF1 gene (NM_000267.1), which spans approximately 350Kb of genomic sequence on chromosome 17q11.2, is composed of 60 exons coding an 8.9Kb mRNA [Cawthon et al., 1990a;Wallace et al., 1990]. NF1 gene product, neurofibromin, is a 250 KDa hydrophilic protein comprising 2818 amino acids [Cawthon et al., 1990b]. The central region of neurofibromin displays marked homology with Ras-GTPase activation proteins (GAPs) that are involved in the negative regulation of RAS-mediated signal transduction pathways [Cichowski and Jacks, 2001].The mutation rate at the NF1 gene is estimated to be ~1x10 -4 /gamete/generation making it one of the highest of any human disease genes [Huson et al., 1989;Upadhyaya and Cooper, 1998]. Mutations encompass both single nucleotide substitutions and large genomic deletions [Stenson et al., 2003]. Approximately a half of all mutations arise de novo and do not appear to be clustered within...

The Arg1038Gly missense variant in the NF1 gene causes a mild phenotype without neurofibromas

“…There are different pieces of evidence supporting the pathogenicity of this variant: it is extremely rare and it is predicted to cause a nonconservative amino acid change, resulting in the replacement of a positively charged arginine with a neutral glycine. This residue lays within a highly conserved region: arginine 1038 is conserved up to arthropods (Figure b) and a different missense substitution affecting the same codon (p.Arg1038Ser) was previously reported in a NF1 patient (Lee et al, ), underlying its relevant role for protein structure and/or function. Most of the prediction software tools (PolyPhen2, SIFT, Mutation Taster) we examined, including those integrating multiple tools such as CADD and REVEL (with score of 30 and 0.661, respectively) are in favor of a pathogenetic role of this substitution.…”

Association of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mutation/Variant/Haplotype and Tumor Necrosis Factor (TNF) Promoter Polymorphism in Hyperlipidemic Pancreatitis

“…Mutations in the PRSS1, SPINK1, and CFTR genes were examined by PCR analysis and then analyzed with denaturing HPLC, according to procedures modified from those described in previous reports (15,25 ). Denaturing HPLC was performed on a WAVE DNA fragment analysis system equipped with a DNASep column and an ultraviolet-C scanner to detect eluted DNA (Transgenomic) (26 ).…”

The Sec14 Homology Module of Neurofibromin Binds Cellular Glycerophospholipids: Mass Spectrometry and Structure of a Lipid Complex

“…16, 18 The physiological importance of Sec14-like domains and their corresponding genes is underscored by the occurrence of mutations associated with human disease, [19][20][21] including NF1. [22][23][24][25][26][27] From a structural point of view Sec14-like domains form a cavity, in which a hydrophobic ligand is accommodated, as seen in Sec14p, 28 SPF 29,30 and αTTP. 31, 32 The Sec14-like module of neurofibromin is neighbor to a PH-like domain, creating thereby an interface, which suggests regulatory features for ligand binding.…”