Silent but deadly: Understanding the importance of ‘silent’ mutations in the Cystic Fibrosis gene


Cystic fibrosis results from the mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene1. This mutation causes decreased chloride ion transport across the surface epithelia and dehydration of the airway surface liquid leading to the accumulation of thick viscous mucus which can trap pathogens setting up chronic bacterial infections.

More than 2,000 mutations in the CFTR gene have been identified1 – approximately 1,700 of which cause cystic fibrosis. An individual will inherit CF if both parents carry a mutation in the CFTR gene. These two faults may be the same or they could consist of two different mutations. The severity of disease varies between individuals and is partly due to how the inherited mutations affect the manufacture of the CFTR protein and how well it works within the cell.

The remaining 270 mutations identified include synonymous single nucleotide polymorphisms (sSNPs) or ‘silent’ mutations2. Due to the degenerative nature of the genetic code – i.e. more than one codon specifies each amino acid, sSNPs were considered to have no effect on the folding and therefore function of the protein produced. It should be noted that sSNPs do not cause disease by themselves, but are more common in patients with the most severe disease. Genome-wide association studies have linked sSNPs with over 50 human diseases so how can such ‘silent’ mutations be having such a detrimental effect?

This question was addressed by several teams, one led by Professor Igatova from the University of Hamburg and Professor Sheppard at the University of Bristol with help from colleagues in the Netherlands and the USA3.

The group at the University of Hamburg identified an sSNP, T2562G, which modifies the local translation speed of the CFTR, leading to detrimental alterations in the protein folding and function3. T2562G is one of the most common SNPs in the CFTR gene, with a prevalence of 34% in the general population4. This sSNP does not cause CF by itself but is it commonly found in patients with CFTR-related disorders5.

Professor Sheppard’s group at the University of Bristol showed that this inaccurate folding caused a narrowing in the final CFTR chloride ion channel which slowed the movement of the ions through the cell membrane3.

T2562G was found to introduce a codon pairing to a low-abundance tRNA which is rare in human bronchial epithelia but interestingly, not in other human tissues. The low abundance of the tRNA coded for by T2562G resulted in a far slower translocation speed at the Thr854 codon leading to vital changes in CFTR stability and function (Fig. 1B). The folding and function of T2562G-CFTR could be rescued by increasing the cellular concentration of the tRNA cognate to the mutant ACG codon (Fig. 1C).

This work illustrates how the function of the CFTR can be influenced by mutations in the CFTR gene that are not in themselves CF-causing. When these ‘silent’ mutations occur in conjunction with CF-causing mutations they can greatly alter the severity of disease. Understanding the effects of these silent changes on protein folding and function will help to understand the root cause of disease and perhaps ultimately find new treatments.

HollyFig. 1

The synonymous single nucleotide polymorphism (sSNP) T2562G inverts local translation speed in CFTR mRNA, which can be rescued by tRNAThr(CGU).

(A) Thr-854–encoding codon ACT in wild-type CFTR is translated fast, as its cognate tRNAThr(AGU) is relatively abundant. (B) T2562G sSNP converts the ACT triplet to ACG codon, which is read by the rare cognate tRNAThr(CGU) and reduces local ribosomal speed. Stochasticity in the delivery of tRNAThr(CGU) cognate to the ACG codon creates variations in the intimate translation speed of each ACG codon at this position and hence generates 2 distinct CFTR channel populations, one with wild-type–like (wtl) CFTR properties and a second with a reduced conductance and a more compact structure (small-conductance [sc] population). (C) Increase of the cellular level of tRNAThr(CGU) pairing to the mutated ACG codon restores ribosome speed at the rare Thr-ACG codon and rescues the CFTR conductance defect.

Blog written by Holly Charlton

References

  1. Cutting GR. Cystic fibrosis genetics: from molecular understanding to clinical application. Nat Rev Genet. 2015;16: 45–56.
  2. Veit G, Avramescu RG, Chiang AN, Houck SA, Cai Z, Peters KW, et al. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations. Mol Biol Cell. 2016;27: 424–33.
  3.  Kirchner S, Cai Z, Rauscher R, Kastelic N, Anding M, Czech A, Kleizen B, Ostedgaard LS, Braakman I, Sheppard DN, Ignatova Z. Alteration of protein function by a silent polymorphism linked to tRNA abundance. PLoS Biol. 2017 May 16;15(5):e2000779.
  4. Cuppens H, Marynen P, De Boeck C, Cassiman JJ. Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene. Genomics. 1993;18: 693–7.
  5. Steiner B, Truninger K, Sanz J, Schaller A, Gallati S. The role of common single-nucleotide polymorphisms on exon 9 and exon 12 skipping in non-mutated CFTR alleles. Human Mutat. 2004;24: 120–9.

 

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