Questions: Missense, Nonsense, and Silent Mutations

Question 1

A geneticist identifies a missense mutation in a patient's BRCA1 gene that changes leucine to isoleucine — a conservative substitution. She tells the patient this change is almost certainly benign. What critical factor is she neglecting?

• A. Whether the mutation was inherited or arose de novo, since de novo mutations are always more severe.
• B. The position of the mutation in the protein — even a conservative substitution at a functionally critical site (active site, binding interface, structurally essential residue) can be pathogenic.
• C. The patient's age at onset, since missense mutations have age-dependent severity.
• D. The overall amino acid composition of BRCA1, since proteins with many leucines tolerate substitutions less well.

Answer: (B)

Chemical similarity (conservative substitution) is only one of three factors determining missense severity. Position in the protein is equally or more critical: a leucine-to-isoleucine change in a disordered or non-functional region is very different from the same change at a BRCA1 domain required for DNA repair or protein-protein interaction. 'Conservative' refers only to chemical similarity — not to functional impact. Evolutionary conservation of a site (whether that position varies across species) is a much better predictor of pathogenicity than substitution type alone.

Question 2

A patient carries a nonsense mutation near the beginning of a gene encoding a transcription factor, and no protein from that allele is detectable. Which mechanism most likely explains the absence of protein?

• A. The truncated protein is produced normally but is rapidly degraded by the proteasome because it lacks its C-terminus.
• B. Nonsense-mediated mRNA decay (NMD) detects the premature stop codon and degrades the mRNA before significant protein can be translated.
• C. The nonsense mutation falls in a region that overlaps the promoter, preventing transcription from initiating.
• D. The ribosome cannot initiate translation when a stop codon is present in the early coding sequence.

Answer: (B)

NMD is a cellular surveillance pathway that recognizes premature termination codons and degrades the mRNA, effectively converting many nonsense mutations into null alleles. For a stop codon near the beginning of the gene (well upstream of the last exon-exon junction), NMD is highly efficient. This prevents the accumulation of potentially toxic truncated proteins and explains why many nonsense mutations phenocopy complete gene deletion. The position still matters: a nonsense mutation near the very end of the coding sequence may escape NMD and produce a nearly full-length protein.

Question 3

A synonymous (silent) mutation — one that does not change the encoded amino acid — can still affect protein expression levels.

• T. True
• F. False

Answer: True

Silent mutations were historically assumed to be truly neutral, but they can affect gene expression through several mechanisms: codon usage bias (rare codons slow ribosome speed, affecting protein folding and expression level), mRNA secondary structure (which affects ribosome processivity and mRNA stability), and exonic splicing enhancers or silencers (some synonymous mutations disrupt splicing regulatory sequences embedded in exons, causing missplicing). These effects are increasingly recognized clinically and challenge the assumption that 'silent' means 'functionally neutral.'

Question 4

The functional impact of a missense mutation is determined primarily by whether the substitution is chemically conservative or non-conservative — conservative substitutions are rarely harmful.

• T. True
• F. False

Answer: False

Position in the protein is as important as chemical similarity. A conservative substitution at a structurally or functionally critical site can be devastating, while a non-conservative substitution in a non-essential disordered region may have no detectable effect. The classic example is sickle cell disease: glutamic acid to valine is a moderate substitution, but its position (surface residue at position 6 of β-globin) allows hemoglobin polymerization under low-oxygen conditions — a severe consequence. Evolutionary conservation of a position (whether it varies across species) is a better predictor of pathogenicity than substitution type alone.

Question 5

Why do evolutionary studies use the ratio of non-synonymous to synonymous substitution rates (dN/dS) to identify functionally constrained regions of genes?

Model answer: Synonymous (silent) mutations don't change the protein sequence, so they are largely neutral and accumulate at a rate reflecting the background mutation rate. Non-synonymous mutations change the protein, so they are subject to natural selection. In functionally constrained regions, almost any amino acid change impairs function, so purifying selection removes non-synonymous variants — making dN/dS < 1. In unconstrained regions, non-synonymous mutations are tolerated and dN/dS ≈ 1. Regions with dN/dS > 1 show positive (adaptive) selection. By comparing substitution rates, researchers can identify which parts of a protein are under selection without knowing the structure — and infer which residues are functionally critical.

The dN/dS ratio is powerful because it uses evolutionary history as a functional assay. Sites conserved across millions of years of evolution are constrained because mutations there are harmful; sites that vary freely tolerate change. This provides a quantitative, genome-wide measure of functional importance that complements structural and biochemical data.