The n e w e ng l a n d j o u r na l of m e dic i n e

Cl inic a l I m pl ic a t ions of B a sic R e se a rch

Elizabeth G. Phimister, Ph.D., Editor

When Silence Disrupts

Ryan C. Hunt, M.D., and Chava Kimchi‑Sarfaty, Ph.D.

A common assumption is that synonymous codon
changes, so called because they do not alter the
encoded amino acid sequence, are neutral genetic
variants that have no effect on phenotype or
genetic fitness. However, there is a mounting
body of literature showing that synonymous
variants influence protein biosynthesis and the
biologic behavior of cells and underlie human
disease. For example, synonymous variants influ-
ence susceptibility to Crohn’s disease,1 promote
the misfolding of the most common mutant
form of the cystic fibrosis transmembrane con-
ductance regulator (CFTR) protein in cystic fi-
brosis,2 and alter the specificity of drug efflux
pumps in cancer cells.3 Disease-associated syn-
onymous variants have been found to affect a
range of cellular processes spanning messenger
RNA (mRNA) and protein synthesis (Fig. 1).2,4-7
Yet, belief in the general neutrality of synony-
mous variants persists, which has ramifications
for population genetics and the interpretation of
genomic results in the clinic. The piecemeal flow
of evidence describing functional synonymous
variants makes it difficult to know how often
nonneutral synonymous variants occur and the
extent of their effects on human biologic pro-
cesses.
Can the nonneutrality of synonymous muta-
tions be proved experimentally and at scale?
Shen and colleagues8 recently sought to address
this question using the model organism Saccha-
romyces cerevisiae (yeast) (Fig. 2). This work was
made possible only by methodologic advances,
namely a gene-editing tool called CRISPR–Cas9
(clustered regularly interspaced short palindromic
repeats associated with Cas9 endonuclease),
which allowed for the systematic creation of
targeted synonymous and nonsynonymous mu-
tations. The authors first created more than
8000 single-nucleotide variants in S. cerevisiae
within the coding regions of 21 nonessential
genes. Each variant was then evaluated for its
effect on S. cerevisiae fitness. Measuring relative
fitness was accomplished by culturing the mu-
tants with wild-type yeast en masse and moni-
toring changes in genotype frequencies (which
reflect the relative changes in mutant vs. wild-
type yeast) over time. With this method, small
absolute differences in fitness among variants
could be reliably measured (Fig. 2).
The median fitness of mutants with nonsense
mutations (which are known to abrogate protein
function) was, as expected, lower than that of
nonsynonymous variants. Surprisingly, three quar-
ters of synonymous variants showed reduced
fitness, and the median fitness of the 1866 syn-
onymous variants was nearly identical to that of
the 6306 nonsynonymous variants. This stun-
ning result directly contradicts assumptions
about the neutrality of synonymous mutations
that have been held for decades. Next, Shen et al.
looked for common mechanisms underlying
observed changes in fitness. They found that
reduced fitness for both synonymous and non-
synonymous variants was most strongly corre-
lated with the relative reduction in mRNA ex-
pression level. This is a somewhat unexpected
finding because nonsynonymous variants are
not thought to commonly exert strong effects on
mRNA levels.
The outcomes of this work have a range of
implications. In addition to challenging assump-
tions central to population genetics, the find-
ings by Shen et al. also call into question the
standard analytic approach to clinical sequenc-
ing. Genomic tests have given clinicians an un-
precedented ability to understand the genetic
basis for human disease, screen for cancer-
driving mutations, diagnose familial cancer syn-
dromes, and justify the use of targeted therapies.

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 The n e w e ng l a n d j o u r na l of m e dic i n e

The Potential Effects of Synonymous Variants

At the Level of mRNA At the Level of Protein

mRNA splicing Initiation and speed of translation

By disrupting or creating consensus splice donor or accepted sites, Specific codons may confer an mRNA structure that permits (or
synonymous variants can lead to aberrant mRNA splicing and mature “discourages”) the initiation of translation of the mRNA into protein.
mRNAs that are unstable or encode flawed proteins. After initiation, the rate of translation can be further affected by mRNA
Premature splice-donor site secondary structure and codon composition because the cellular
pools (bioavailability) of specific cognate tRNAs vary among
5'UTR Exon 1 Exon 3 Exon 4 3'UTR synonymous codons.
TTP
mRNA encoding TTP
5'UTR Exon 1 Exon 3 Exon 4 3'UTR Prematurely truncated Reduced levels
protein of TTP
Example: Synonymous variant
Example:
A synonymous mutation in MECP2 creates a premature splice-donor
A synonymous variant hampers translation of the tumor suppressor
site. This results in a truncated MECP2 protein and Rett’s syndrome.5
TTP and is associated with response of breast cancer to trastuzumab.6

mRNA structure and stability

Stem−loops and helices in the mRNA secondary structure can be Co-translational protein folding
created or “eradicated” by synonymous codon changes. Altering the This mechanism is secondary to the effect on speed of translation.
structure of mRNA affects the synthesis of proteins by altering Rare versus common synonymous codons can influence the kinetics
stability or half-life of mRNA transcript, ribosome accessibility, and of co-translational protein folding and access to protein-folding
translational efficiency. machinery. Altogether, this can result in a protein with altered
Nonvariant CFTR ∆F508 CFTR
conformation.
(IIe507ATT) (IIe507ATC) mRNA encoding tRNA
ATT ATC MDR1
5' Common codons Rare codons Misfolded
mRNA protein
fragment Fast Slow Fast
Example: Example:
The most common pathogenic mutation in CFTR (causing cystic A synonymous mutation in MDR1 alters the substrate specificity of the
fibrosis) results in a synonymous codon at position 507, which transporter by altering protein conformation.3
changes the secondary structure of the mRNA. This modified
structure is associated with misfolded and reduced levels of CFTR
Post-translational modifications
protein.2
Effects on post-translational modifications (such as phosphorylation)
can occur when translational speed and protein folding are altered by
MicroRNA binding synonymous codons.
MicroRNAs regulate gene expression through binding mRNAs and mRNA
mRNA encoding β-actin mRNA encoding γ-actin
-actin
mediating their degradation (or blocking translation). Their binding sequences differ by
sites in mRNAs can be disrupted by synonymous codon changes 13% at the nucleotide level
Slow translation
(red highlight below). owing to synonymous changes
Normal Disrupted
binding site binding site Protein amino acid Ub
IRGM c.313C 5' C CC G GA G 3' U G 3' β-actin sequences are nearly γ-actin
-actin
U• C• A• A• C• U
• C• A• A• C• U
• A• C• C• U
• • A• C• U
A• C• U • identical
AGUUG AG U UGA UGG A UGA UG A
miRNA-196A 3' GGGU U U U 5' G U 5' Example:
β- and γ-actin have different and consequential post-translational
β-
Example: modifications despite being almost identical at the amino acid level.
By disrupting the consensus binding site of a microRNA, a single Synonymous substitutions in γ-actin mRNA result in slower
synonymous variant in IRGM contributes to Crohn’s disease translation, exposing a normally inaccessible lysine residue that is
susceptibility through dysregulated xenophagy.1 consequently ubiquitinated; the modified γ-actin is degraded.7

Figure 1. The Potential Effects of Synonymous Variants.

Although synonymous (or “silent”) variants have no effect on amino acid sequence, some synonymous variants influence fitness and
susceptibility to disease. Mechanisms include interference with messenger RNA (mRNA) splicing, structure, and stability and disruption
of microRNA binding to mRNA. They also include changes in the speed of translation and thus access to the protein‑folding machinery
and enzymes that chemically modify proteins. The abbreviation tRNAs denotes transfer RNAs, Ub ubiquitin, and UTR untranslated region.

Clinicians are accustomed to receiving a se- However, an overlooked aspect of clinical

quencing report with an annotated list of vari- genetics is the bioinformatic workflow for iden-
ants categorized on a scale of pathogenic like- tifying and ascribing pathogenicity (or a benign
lihood. status) to genetic variants. Exome and genome

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 Clinical Implications of Basic Research

Testing the Effect of Synonymous Variants

Results showed 0.25 Nonsynonymous

Saccharomyces cerevisiae 5 nearly identical fitness

Fraction of Variants with

Synonymous

a Specific Fitness Level

1 between synonymous 0.20
and nonsynonymous
variants 0.15

0.10

Introduction of variants 0.05

(one per yeast cell) by
Cas9/gRNA 0.00
0.8 0.9 1.0 1.1
2 Fitness

Nonsynonymous Wild type

variants
Synonymous Measure of fitness derived from
variants relative numbers of sequence
4
reads of variant and
wild-type yeast

Yeast compete for

More than 8000 variants, each growth
harbored by a yeast cell with a different Equivalent representation of sequence
single-nucleotide variant in 1 of 21 genes reads indicates similar levels of the different
yeast strains in growth medium

Figure 2. Testing the Effect of Synonymous Variants.

In a recent study, Shen et al.8 systematically created more than 8000 unique single‑nucleotide variants in 21 nonessential coding genes
in the model organism Saccharomyces cerevisiae. Next, they carried out competitive growth experiments, whereby all variants of a gene
were cultured en masse. After coculture, the genotype frequencies of each variant — a sensitive measurement of the relative growth —
were determined by means of next‑generation sequencing. The effects on fitness for the 6306 nonsynonymous and 1866 synonymous
variants were nearly identical. The systematic methodologic approaches used by Shen et al. made it possible to prove at scale the perva‑
sive effect of functional synonymous variants. Experiments such as the one described by Shen et al. should motivate further studies to
assess how synonymous variants influence protein expression and function and the extent to which they are relevant to human disease.
The abbreviation gRNA denotes guide RNA.

sequencing can generate millions of variants
that need to be filtered down to those likely to
be medically meaningful. Guidelines from the
American College of Medical Genetics and Ge-
nomics state that synonymous variants — un-
less predicted to disrupt splicing — should be
classified as “likely benign.”9 Synonymous vari-
ants are therefore excluded early in bioinfor-
matic pipelines and not evaluated as potential
causes of disease. Certainly, our ability to better
evaluate synonymous variants will hinge on the
development of reliable computational tools to
holistically assess them. Such tools might take
into account the sequence context of the variant
and knowledge about how the variant could af-
fect the interaction between mRNA and the ribo-
some during protein synthesis.
The work by Shen et al. also ties into the
development of recombinant therapeutic pro-
teins. Most modern-day therapeutic proteins are
the product of recombinant protein engineering.
Modifying the native protein can allow for en-
hanced function or a prolonged half-life. The
expression sequence of the protein can also be
revised with synonymous codon substitutions to
ease cloning or to achieve heightened protein
expression in a host cell. The ability to modulate
therapeutic proteins has expanded rapidly, but
predicting when and how synonymous substitu-
tions affect protein structure and the risk of
immunogenicity remains a challenge. Large-
scale experiments such as the one described by
Shen et al. should motivate further studies to
assess how synonymous variants influence pro-

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 Clinical Implications of Basic Research

tein expression and function and the extent to
which they are relevant to human disease.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
From the Division of Plasma Protein Therapeutics, Office of
Tissues and Advanced Therapies, Center for Biologics Evalua‑
tion and Research, Food and Drug Administration, Silver
Spring, MD.
1. Brest P, Lapaquette P, Souidi M, et al. A synonymous variant
in IRGM alters a binding site for miR-196 and causes deregula-
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2. Bartoszewski RA, Jablonsky M, Bartoszewska S, et al. A syn-
onymous single nucleotide polymorphism in DeltaF508 CFTR
alters the secondary structure of the mRNA and the expression
of the mutant protein. J Biol Chem 2010;​285:​28741-8.
3. Kimchi-Sarfaty C, Oh JM, Kim I-W, et al. A “silent” polymor-
phism in the MDR1 gene changes substrate specificity. Science
2007;​315:​525-8.
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morphism of the Tristetraprolin (TTP) gene, an AU-rich mRNA-
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DOI: 10.1056/NEJMcibr2207405
Copyright © 2022 Massachusetts Medical Society.

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