A novel NGF mutation clarifies the molecular mechanism and extends the phenotypic spectrum of the HSAN5 neuropathy

Abstract

Background: Nerve growth factor β (NGFβ) and tyrosine kinase receptor type A (TRKA) are a well studied neurotrophin/receptor duo involved in neuronal survival and differentiation. The only previously reported hereditary sensory neuropathy caused by an NGF mutation, c.661C>T (HSAN5), and the pathology caused by biallelic mutations in the TRKA gene (NTRK1) (HSAN4), share only some clinical features. A consanguineous Arab family, where five of the six children were completely unable to perceive pain, were mentally retarded, did not sweat, could not discriminate temperature, and had a chronic immunodeficiency, is reported here. The condition is linked to a new homozygous mutation in the NGF gene, c.[680C>A]+[681_682delGG].

Methods: Genetic linkage and standard sequencing techniques were used to identify the causative gene. Using wild-type or mutant over-expression constructs transfected into PC12 and COS-7 cells, the cellular and molecular consequences of the mutations were investigated.

Results: The mutant gene produced a precursor protein V232fs that was unable to differentiate PC12 cells. V232fs was not secreted from cells as mature NGFβ.

Conclusions: Both the clinical and cellular data suggest that the c.[680C>A]+[681_682delGG] NGF mutation is a functional null. The HSAN5 phenotype is extended to encompass HSAN4-like characteristics. It is concluded that the HSAN4 and HSAN5 phenotypes are parts of a phenotypic spectrum caused by changes in the NGF/TRKA signalling pathway.

Figure 1

Clinical phenotype and identification of the mutation. (A) Pedigree of the family showing relationship between affected and unaffected members. Clinical features of family members with NGF c.[680C>A]+[681_682delGG] mutation (CAdGG), showing damage to digits caused by biting (B), traumatic loss of tip of tongue and loss of teeth due to gingivitis(C), and a painless dislocation of the elbow joint (D). (E) Chromatograms comparing the wild-type carrier sequence (top) with that from an affected child (middle), and an alignment of the two sequences showing the altered base and two base pair deletion (bottom). (F) Alignment of part of the mature protein sequence showing the predicted altered amino acid sequence for the protein produced in the previously reported family (R221W) and in our family (V232fs) compared to wild-type NGF.

Figure 2

V232fs and R221W fail to induce PC12 differentiation and are not secreted from the cell. (A) PC12 cells transfected with empty vector (pIRES) or vector containing wild-type (WT), CT (R221W) or CAdGG (V232fs) mutant NGF genes. (B) Cells were monitored over a 72 h time course, and the percentage of differentiated cells was assessed at 24 h intervals. Images showing the relative differentiation of cells expressing WT (top left), R221W (bottom left), V232fs (bottom right) proteins, and empty vector control (top right) are shown. Amplification ×100. (C) Graph showing the percentage of differentiated cells for each of the proteins under study over 3 days. Two-way analysis of variance (ANOVA) analysis found significant differences between WT and both mutants at all the time points (p<0.01). There was no consistent difference between both mutants. Error bars represent mean ±SEM. COS-7 cells were transfected with empty vector or a vector containing wild-type, or either of the mutant (CT and CAdGG) NGF genes. 48 h after transfection, the media from the cells expressing WT, R221W or V232fs NGF was collected and concentrated, and cells were lysed. Total protein concentration was measured for each sample, before being assayed for NGF by ELISA. Results shown are the average of two independent experiments. Analysis with a one-way repeated measures ANOVA found NGF levels in wild-type were significantly higher than in either mutant for both the cellular extracts and media (p<0.01).

Figure 3

V232fs is not processed while R221W is processed poorly. To determine whether the levels detected by ELISA represented precursor NGF or mature NGFβ, extracts were subjected to SDS PAGE, transferred to PVDF and the membranes probed with anti-NGF antibody to detect both precursor and mature forms (top), anti-FLAG specific for precursor form only (middle), and β-actin as a loading control (bottom). Both mutants seemed to fail to complete processing, although a faint band can be detected for the CT mutant in the media extract.