Quantitative sequence analysis ofFBN1premature termination codons provides evidence for incomplete NMD in leukocytes

Abstract

We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.

High prevalence of germline STK11mutations in Hungarian Peutz-Jeghers Syndrome patients

“…Despite the semi-quantitative nature of this method, our results are similar to those obtained by other groups examining mutated alleles of several different genes [42,43]. The reduction of the level of premature stop codon-containing transcripts is partially attributable to the mechanism of nonsense-mediated mRNA-decay, an evolutionarily conserved mechanism distinguishing normal from premature termination codons on the basis of their location with respect to exon-exon junctions, and targeting the latter for degradation [44].…”

Analysis of 31-year-old patient with SYNGAP1 gene defect points to importance of variants in broader splice regions and reveals developmental trajectory of SYNGAP1-associated phenotype: case report

“…Therefore we could observe a total of three transcripts with predicted premature termination codons, one in normal controls and three in the patient. As our experimental conditions did not block nonsense-mediated RNA decay (NMD) [9, 10], the transcripts seem not to be subject of NMD. This may reflect the fact that the efficiency of NMD in lymphocytes is lower than in other cell types [10].…”

“…As our experimental conditions did not block nonsense-mediated RNA decay (NMD) [9, 10], the transcripts seem not to be subject of NMD. This may reflect the fact that the efficiency of NMD in lymphocytes is lower than in other cell types [10]. It has to be stressed that our experiment using peripheral blood lymphocyte mRNA may not fully reflect the expression of SYNGAP1 in the brain.…”

Hungarian Marfan family with large FBN1 deletion calls attention to copy number variation detection in the current NGS era

“…At protein level, the deletion of coding exons 2-4 is predicted to lead to a frameshift and a premature termination codon [NP_000129.3:p.(Pro56Cysfs*3)]. Thus, nonsense mediated mRNA decay may take place resulting in functional haploinsufficiency (15). Testing of the index patient's firstdegree relatives for the detected FBN1 deletion by MLPA and Sanger sequencing revealed that the deletion is present in the patient's sister and mother but absent in the father, as expected based on clinical features (Figure 2).…”