Nonsense-mediated mRNA decay process in nine alleles of Niemann-Pick type C patients from Spain
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.
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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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Pre-mRNA splicing defects and RNA binding protein involvement in Niemann Pick type C disease
2020, Journal of BiotechnologyCitation Excerpt :Degradation via NMD of NPC1 transcripts bearing premature stop codons generated by nine nonsense or frame-shift mutations and one deletion has been demonstrated. In all cases the mRNA (quantified with qPCR) and the protein were not detectable, compared to controls (Macías-Vidal et al., 2009; Tamura et al., 2006). Furthermore, a pathological cooperation between splicing defects and mRNA NMD can be established: mutations in splicing regulatory element can produce an abnormal transcript leading to the shifting of the reading frame with the consequent creation of a premature stop codon (Fig. 3B).
Diagnostic tests for Niemann-Pick disease type C (NP-C): A critical review
2016, Molecular Genetics and MetabolismCitation Excerpt :cDNA sequencing is helpful to detect intronic problems and for identification of splice mutations; several early NPC1 mutational studies utilised cDNA sequencing [28,115,116], and in some instances revealed the effect of complex genomic mutations. Multiplex ligation-dependent probe amplification (MLPA) and quantitative PCR can be used to detect genomic rearrangements (exonic or whole gene deletions) [10,108,113,117], and assessment of mRNA degradation (inhibition of nonsense-mediated mRNA decay processes) has also been used to investigate mutations resulting in aberrant splicing [10,25,118]. NGS methodologies can be used for gene panel sequencing, large-scale genetic analysis, and whole exome and whole genome analyses, and have recently been employed in NP-C screening and diagnosis.
A leaky splicing mutation in NFU1 is associated with a particular biochemical phenotype. Consequences for the diagnosis
2016, MitochondrionCitation Excerpt :Single-stranded cDNA was obtained using oligo-dT primers and M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant (Promega, Madison, WI, USA) according to the manufacturer's protocol. To analyze the transcript generated by the c.545 + 5G > A splice site mutation cells were treated with cycloheximide (CHX) (Sigma, St. Louis, MO) according to the protocol previously described (Macías-Vidal et al., 2009). NFU1 cDNA was amplified by polymerase chain reaction (PCR) using self-designed oligonucleotides (available upon request) and analyzed by Sanger sequencing.
The early detection of Salla disease through second-tier tests in newborn screening: How to face incidental findings
2014, European Journal of Medical GeneticsCitation Excerpt :The content of free sialic acid was then quantified using a spectrophotometer at 580 nm [Tsai and Marshall, 1979]. After informed consent was received from the parents, a skin biopsy was obtained from the patient and fibroblasts were cultured as described [Macías-Vidal et al., 2009]. To detect possible mutations whose mRNAs are candidate to suffer nonsense-mediated mRNA decay (NMD) process, fibroblasts were treated with cycloheximide (CHX) (Sigma, St. Louis, MO) according to the protocol previously described [Macías-Vidal et al., 2009].