Nonsense-mediated mRNA decay process in nine alleles of Niemann-Pick type C patients from Spain

https://doi.org/10.1016/j.ymgme.2009.01.007Get rights and content

Abstract

Mutations in NPC1 or NPC2 genes are responsible of Niemann-Pick type C disease (OMIM #257220), an autosomal recessive neurodegenerative lysosomal storage disorder caused by a non-regulation of intracellular lipid trafficking.

Alterations such as nonsense or frame shift mutations generate a premature termination-codon (PTC). Nonsense-mediated mRNA decay (NMD) is a natural cellular process that degrades mRNAs that encode a prematurely truncated protein.

In this study we have analyzed 9 NPC1 mutations which generate a PTC (p.R116X, p.Q119VfsX8, p.W260X, p.S425X, p.A558GfsX12, p.Q775X, p.G993EfsX4, p.R1059X and p.I1061NfsX4), in order to determine if their mRNAs suffer NMD process. To achieve this objective we compared fibroblasts of patients carrying these alleles with and without cycloheximide (CHX) treatment using conventional PCR and real-time PCR.

The results of conventional PCR of untreated fibroblasts showed a reduction of the amount of NPC1 mRNA compared to control in all patients. After CHX-treatment, a recovery of mRNA was detected but not in all the alleles. However, when real-time PCR was used, the recovery was observed including those alleles that qualitatively showed no apparent increase in mRNA level. In conclusion, we confirmed that NMD process is responsible for the mRNA decay for all the analyzed NPC1 PTC-encoding mutations.

Introduction

Mutations in the NPC1 (RefSeq NM_000271.3) [1] (95%) and NPC2 (RefSeq NM_006432.3) [1] (5%) genes are responsible for Niemann-Pick type C disease (OMIM #257220) [2], a neurodegenerative autosomal recessive lysosomal disorder. Over 270 different mutations have been described worldwide in NPC1 gene [3], which is located in 18q11-q12 chromosome [4]. It spans 47 Kb, has 25 exons and encodes a NPC1 transmembrane protein composed by 1278 amino acids. Although most of the pathogenic described alterations are missense, other types of mutations, which generate a premature termination-codon (PTC) such as nonsense and frame shift, have also been reported. It is known that these kinds of mutations may cause mRNA degradation by a post-transcriptional mechanism called nonsense-mediated mRNA decay (NMD). NMD is used by eukaryotic cells to control the quality of the mRNA in order to prevent the expansion of truncated polypeptides [5]. It has been described that NMD process takes place when PTC occurs more than 50–55 nucleotides upstream of the 3′-most exon–exon junction [6]. The protein synthesis inhibitor cycloheximide (CHX) is known to suppress NMD [7] and therefore, is used as proof of NMD pathway involvement.

In this study, we wanted to determine whether NMD mechanism affected the stability of NPC1 mRNAs bearing a PTC. For this purpose, we performed the analysis of 9 selected nonsense or frame shift NPC1 mutations. We analyzed and compared the results obtained using conventional PCR and real-time PCR.

Section snippets

Patients and controls

Samples were obtained from 11 unrelated patients. All of them are of Spanish origin except two patients, one Moroccan (NPC41) and one Portuguese (NPC42). Diagnosis of NPC disease was determined by cytochemical demonstration of pathologically enriched cholesterol via filipin staining [8].

The control group consisted of four samples from non-related wild-type subjects. To perform the experiments, we prepared a pool of control cDNAs.

Cell culture

Fibroblasts from skin biopsies of control individuals and patients

Results

Table 1 reports NPC1 gene mutations of the 11 unrelated patients of the present study. Among the identified changes, there are 3 novel mutations (p.G535V (c.1604G>T), p.Q775X (c.2323C>T) and p.I1061NfsX4 (c.3181dupA)) and 11 previously described, 6 of which by our group [10].

The suspicion that NMD could degrade the mRNA encoded by these mutations was given by NPC20 patient. In this case, p.I1061T mutation was found in homozygosis in the cDNA, while actually, this mutation was observed in

Discussion

In mammals, the process of nonsense-mediated mRNA decay (NMD) is a quality-control mechanism to degrade mRNA harboring a premature termination-codon to prevent the synthesis of truncated proteins [5].

Here, we have presented 11 unrelated NPC patients, all of them bearing at least one PTC-encoding mutation in their NPC1 gene.

From the series presented, we have described 3 novel mutations, two generating PTC (p.Q775X and p.I1061NfsX4) and one novel missense change (p.G535V). This last mutation is

Acknowledgements

This research was supported by a Grant from the Fundación Niemann-Pick de España. The authors are grateful to them and also to all the patients and their families. J. Macías-Vidal is recipient of Juan Girón fellowship from the Spanish Foundation.

We thank all the physicians who referred patients for the study: Dr. Ruiz del Portal (Hospital Virgen del Rocío), Dr. Verdú (Hospital Virgen de la Salud), Dr. Jara (Hospital Universitario La Paz), Dr. Casas (Hospital Virgen de la Arrixaca), Dr. Sánchez

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