Qualitative and quantitative analysis of FBN1 mRNA from 16 patients with Marfan Syndrome
Lena Tjeldhorn1, Silja Svanstrøm Amundsen2,3, Tuva Barøy4,5
1Department of Medical Genetics, Oslo University Hospital, Box 4950, 0424, Oslo, Norway. lena.tjeldhorn@online.no.
Background:
Pathogenic mutations in FBN1, encoding the glycoprotein, fibrillin-1, cause Marfan syndrome (MFS) and related connective tissue disorders. In the present study, qualitative and quantitative effects of 16 mutations, identified in FBN1 in MFS patients with systematically described phenotypes, were investigated in vitro.
Methods:
Qualitative analysis was performed with reverse transcription-PCR (RT-PCR) and gel electrophoresis, and quantitative analysis to determine the FBN1 mRNA levels in fibroblasts from the 16 patients with MFS was performed with real-time PCR.
Results:
Qualitative analysis documented that the mutations c.4817-2delA and c.A4925G led to aberrant FBN1 mRNA splicing leading to in frame deletion of exon 39 and in exon 39, respectively. No difference in the mean FBN1 mRNA level was observed between the entire group of cases and controls, nor between the group of patients with missense mutations and controls. The mean expression levels associated with premature termination codon (PTC) and splice site mutations were significantly lower than the levels in patients with missense mutations. A high level of FBN1 mRNA in the patient with the missense mutation c.G2447T did not segregate with the mutation in three of his first degree relatives. No association was indicated between the FBN1 transcript level and specific phenotypic manifestations.
Conclusions:
Abnormal FBN1 transcripts were indicated in fibroblasts from patients with the splice site mutation c.4817-2delA and the missense mutation c.A4925G. While the mean FBN1 mRNA expression level in fibroblasts from patients with splice site and PTC mutations were lower than the mean level in patients with missense mutations and controls, inter-individual variability was high. The observation that high level of FBN1 mRNA in the patient with the missense mutation c.G2447T did not segregate with the mutation in the family suggests that variable expression of the normal FBN1 allele may contribute to explain the variability in FBN1 mRNA level.
Insights
Pathogenic mutations in FBN1 cause Marfan syndrome (MFS). This study found abnormal FBN1 mRNA splicing in some MFS patients, with lower expression for splice site and PTC mutations, but high variability.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Pathogenic mutations in FBN1, encoding fibrillin-1, are the primary cause of Marfan syndrome (MFS) and related connective tissue disorders.
- Understanding the in vitro effects of specific FBN1 mutations is crucial for elucidating disease mechanisms.
- This study investigates 16 FBN1 mutations identified in MFS patients with well-defined phenotypes.
Purpose of the Study:
- To qualitatively and quantitatively analyze the effects of 16 identified FBN1 mutations on FBN1 mRNA.
- To correlate FBN1 mRNA levels and splicing abnormalities with MFS phenotypes.
- To investigate the impact of different mutation types (splice site, missense, PTC) on FBN1 expression.
Main Methods:
- Qualitative analysis of FBN1 mRNA splicing using reverse transcription-PCR (RT-PCR) and gel electrophoresis.
- Quantitative analysis of FBN1 mRNA levels in patient-derived fibroblasts using real-time PCR.
- Comparison of FBN1 mRNA levels between MFS patients and healthy controls, and across different mutation types.
Main Results:
- Aberrant FBN1 mRNA splicing was observed for mutations c.4817-2delA and c.A4925G, leading to exon deletions.
- No significant difference in mean FBN1 mRNA levels was found between all MFS patients and controls, or between missense mutation patients and controls.
- Mean FBN1 mRNA levels were significantly lower in patients with premature termination codon (PTC) and splice site mutations compared to missense mutations. High FBN1 mRNA in one patient with a missense mutation did not segregate with the mutation in relatives.
Conclusions:
- Specific FBN1 mutations (c.4817-2delA, c.A4925G) result in abnormal FBN1 transcripts.
- While splice site and PTC mutations show lower mean FBN1 mRNA levels, significant inter-individual variability exists.
- Variable expression of the normal FBN1 allele may contribute to the observed variability in FBN1 mRNA levels, independent of mutation type.
