In genetics, a missense mutation is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid.^[1] It is a type of nonsynonymous substitution.

Missense mutation refers to a change in one amino acid in a protein arising from a point mutation in a single nucleotide. Amino acid's are the building blocks of proteins. Missense mutations are a type of nonsynonymous substitution in a DNA sequence. Two other types of nonsynonymous substitutions are the nonsense mutations, in which a codon is changed to a premature stop codon that results in the resulting protein being cut short, and the nonstop mutations, in which a stop codon deletion results in a longer but nonfunctional protein.

Missense mutations can render the resulting protein nonfunctional,^[2] due to misfolding of the protein,^[3], and these mutations are responsible for human diseases such as Epidermolysis bullosa,^[4] sickle-cell disease^[5] SOD1 mediated ALS, and a substantial number of cancers.^[6][7]

Not all missense mutations lead to appreciable protein changes. An amino acid may be replaced by an amino acid of very similar chemical properties, in which case, the protein may still function normally; this is termed a neutral, "quiet", "silent" or conservative mutation. Alternatively, the amino acid substitution could occur in a region of the protein which does not significantly affect the protein secondary structure or function. When an amino acid may be encoded by more than one codon (so-called "degenerate coding") a mutation in a codon may not produce any change in translation; this would be a synonymous substitution and not a missense mutation.

    DNA: 5' - AAC AGC CTG CGT ACG GCT CTC - 3'
         3' - TTG TCG GAC GCA TGC CGA GAG - 5'
   mRNA: 5' - AAC AGC CUG CGU ACG GCU CUC - 3'
Protein:      Asn Ser Leu Arg Thr Ala Leu

LMNA missense mutation (c.1580G>T) introduced at LMNA gene – position 1580 (nt) in the DNA sequence (CGT) causing the guanine to be replaced with the thymine, yielding CTT in the DNA sequence. This results at the protein level in the replacement of the arginine by the leucine at the position 527.^[8] This leads to destruction of salt bridge and structure destabilization. At phenotype level this manifests with overlapping mandibuloacral dysplasia and progeria syndrome.

The resulting transcript and protein product is:

    DNA: 5' - AAC AGC CTG CTT ACG GCT CTC - 3'
         3' - TTG TCG GAC GAA TGC CGA GAG - 5'
   mRNA: 5' - AAC AGC CUG CUU ACG GCU CUC - 3'
Protein:      Asn Ser Leu Leu Thr Ala Leu

Missense mutations in the MeCP2 protein can cause Rett syndrome, otherwise known as the RTT phenotype.^[9] T158M, R306C and R133C are the most common missense mutations causing RTT.^[9] T158M is a mutation of an adenine being substituted for a guanine causing the threonine at amino acid position 158 being substituted with a methionine.^[10] R133C is a mutation of a cytosine at base position 417 in the gene encoding the MeCP2 protein being substituted for a thymine, causing an amino acid substitution at position 133 in the protein of arginine with cysteine.^[11]

In the most common variant of sickle-cell disease, the 20th nucleotide of the gene for the beta chain of hemoglobin is altered from the codon GAG to GTG. Thus, the 6th amino acid glutamic acid is substituted by valine—notated as an "E6V" mutation—and the protein is sufficiently altered to cause the sickle-cell disease.^[12]

Next Generation Sequencing (NGS) utilizes massively parallel sequencing to sequence the genome. This involves clonally amplified DNA fragments that can be spatially separated into second generation sequencing (SGS) or third generation sequencing (TGS) platforms.^[13] Using massively parallel sequencing allows the NGS platform to produce very large sequences in a single run.^[14] The DNA fragments are typically separated by length using gel electrophoresis.

NGS consists of four main steps, DNA isolation, target enrichment, sequencing, and data analysis.^[14] The DNA isolation step involves breaking the genomic DNA into many small fragments. There are many different mechanisms that can be used to accomplish this such as mechanical methods, enzymatic digestion, and more.^[15] This step also comprises of adding adaptors to either end of the DNA fragments that are complementary to the flow cell oligos and include primer binding sites for the target DNA. The target enrichment step amplifies the region of interest. This includes creating a complementary strand to the DNA fragments through hybridization to a flow cell oligo. It then gets denatured and bridge amplification occurs before the reverse strand is finally washed and sequencing can occur. The sequencing step involves massive parallel sequencing of all DNA fragments simultaneously using a NGS sequencer. This information is saved and analyzed in the last step, data analysis, using bioinformatics software. This compares the sequences to a reference genome to align the fragments and show mutations in the targeted area of the sequence.^[15]

Newborn screening (NBS) for missense mutations is increasingly incorporating genomic technologies in addition to traditional biochemical methods to improve the detection of genetic disorders early in life. Traditional NBS primarily relies on biochemical assays, such as tandem mass spectrometry, to detect metabolic abnormalities indicative of conditions like phenylketonuria or congenital hypothyroidism. However, these methods may miss genetic causes or produce ambiguous results. To address these deficiencies, next-generation sequencing (NGS) is being added to NBS programs. For instance, targeted gene panels and whole-exome sequencing (WES) are used to identify disease causing missense mutations in genes associated with treatable conditions, such as severe combined immunodeficiency (SCID) and cystic fibrosis. Studies like the BabyDetect project have demonstrated the utility of genomic screening in identifying disorders missed by conventional methods, with actionable results for conditions affecting more than 400 genes[3][4][6].^[16][17] In addition, genomic approaches allow for the detection of rare or recessive conditions that may not manifest biochemically at birth, significantly expanding the scope of diseases screened[5][6].^[18] These advancements align with the established principles of NBS, which emphasize early detection and intervention to prevent morbidity and mortality.^[19]

Cancer associated missense mutations can lead to drastic destabilisation of the resulting protein.^[20] A method to screen for such changes was proposed in 2012, namely fast parallel proteolysis (FASTpp).^[21]

DNA polymerases, used in DNA replication, have a high specificity of 10⁴ to 10⁶-fold, in base pairing.^[22] In the base mismatches that occur, 90 to 99.9% are repaired by the DNA mismatch repair pathway, inherent in cells.^[22][23][24] The DNA mismatch repair pathway uses exonucleases, that move along the DNA strand and remove the incorrectly incorporated base, in order for DNA polymerase to fill in the correct base. Exonuclease1 is involved in many DNA repair systems and moves 5' to 3' on the DNA strand.^[25]

More recently, research has explored the use of genetic engineering^[26] and pharmaceuticals as potential treatments.^[27][28] tRNA therapies have emerged in research studies as a potential missense mutation treatment, following evidence supporting their use in nonsense mutation correction.^[29] Missense-correcting tRNAs are engineered to identify the mutated codon, but carry the correct charged amino acid, which is inserted into the nascent protein.^[26] Pharmaceuticals that target specific proteins affected by missense mutations, have also shown therapeutic potential.^[27][28] Pharmaceutical studies have particularly focused on targeting the p53 mutant protein and Ca^2+ channel abnormalities, both caused by gain of function missense mutations, due to their high prevalence in a number of cancers and genetic diseases respectively.^[28][29]

• Ka/Ks ratio
• Missense mRNA