MSH6 Human

What is the MSH6 gene and what is its function in human cells?

The MSH6 (mutS homolog 6) gene is a critical component of the DNA mismatch repair (MMR) system in human cells. Located on chromosome 2, the gene spans 24 kb of genomic sequence and consists of 10 exons, encoding a 1360 amino acid protein . MSH6 functions exclusively as a heterodimer with MSH2, forming the protein complex known as hMutSα (hMSH2-hMSH6) . This complex plays a crucial role in recognizing single base mismatches and small insertion/deletion loops in DNA.

The recognition process is regulated by ADP to ATP transformation, with the Walker-A/B adenine nucleotide binding motif in MSH6 being the most highly conserved sequence found in all MutS homologs . Failure of this repair mechanism results in microsatellite instability and an elevated spontaneous mutation rate, known as a mutator phenotype .

While the MMR pathway is highly conserved from prokaryotes to eukaryotes, the human MSH6 protein shares only 24% amino acid identity with the E. coli version in conserved regions, and notably, the N-terminal of the human MSH6 protein is extended by 400 amino acids compared to its bacterial counterpart .

What are the structural domains of the MSH6 protein and their functions?

MSH6 is a complex protein with five functional domains that work in concert to facilitate DNA mismatch repair:

The MSH6 protein is pseudosymmetric with MSH2, sharing a similar domain structure . This structural relationship is essential for the formation of the functional heterodimer hMutSα. Additionally, human MSH6 has an extended N-terminal region of approximately 400 amino acids compared to prokaryotic versions, whose precise function remains an area of active investigation .

What methods are used to detect MSH6 mutations in clinical and research settings?

Researchers employ multiple complementary techniques to detect and characterize MSH6 mutations:

• Denaturing Gradient-Gel Electrophoresis (DGGE): Commonly used for mutation screening in DNA from peripheral blood lymphocytes, with variants confirmed by direct sequencing of independently amplified PCR products .

• Direct Sequencing: Provides definitive nucleotide sequence identification, typically performed with automated DNA sequencers such as the ABI PRISM 377 .

• Microsatellite Instability (MSI) Analysis: While not directly detecting MSH6 mutations, MSI analysis indicates defective mismatch repair. For MSH6 mutation carriers, MSI is primarily observed at mononucleotide markers rather than dinucleotide repeats . The standard panel includes:

  • Mononucleotide repeats: BAT25, BAT26, and often BAT40 (specifically included for MSH6 analysis)

  • Dinucleotide repeats: D2S123, D5S346, and D17S250

• Immunohistochemical Analysis: Assesses expression of MLH1, MSH2, and MSH6 proteins in tumor tissue. Loss of MSH6 staining can indicate the presence of an MSH6 mutation, though not all tumors from MSH6 mutation carriers show loss of staining .

• Functional Assays: For variants of uncertain significance, in vitro assays using circular DNA substrates with mismatches can measure MMR activity of variant proteins .

Notably, 54% of colorectal and endometrial cancers from MSH6 mutation carriers showed no or only weak MSI , suggesting that MSI alone should not be a definitive selection criterion for MSH6-mutation analysis.

How do MSH6 mutations differ from other mismatch repair gene mutations in their phenotypic manifestations?

MSH6 mutations produce a distinctive clinical phenotype compared to other MMR gene mutations:

This distinctive phenotype has important implications for clinical identification of MSH6 mutation carriers, as traditional Lynch syndrome diagnostic approaches developed based on MLH1/MSH2 mutations may miss many MSH6 carriers. Researchers now recommend considering MSH6-mutation analysis in all patients suspected to have hereditary nonpolyposis colorectal cancer (HNPCC), regardless of MSI or immunohistochemistry results .

What approaches are used to classify MSH6 variants of uncertain significance (VUS)?

The classification of MSH6 variants of uncertain significance requires integration of multiple evidence types:

Interestingly, clinical data from one study revealed "a great resemblance between missense-variant carriers and truncating-mutation carriers," suggesting that many missense variants labeled as "doubtfully pathogenic" based solely on molecular criteria may indeed be pathogenic when clinical features are considered .

How do functional assays contribute to the determination of MSH6 variant pathogenicity?

Functional assays provide critical experimental evidence for MSH6 variant classification according to the ACMG/AMP PS3/BS3 criterion . The primary MMR activity assay methodology involves:

• Preparation of a circular DNA substrate containing:

  • A base:base mismatch or small insertion in an endonuclease recognition site

  • A single-stranded nick to direct the repair process

• Testing environment containing:

  • Variant MSH6 protein supplemented with wild-type MSH2

  • Cellular extract from an MSH6-deficient cell line

• Analysis process:

  • Mismatch correction regenerates a cleavage site

  • Quantification of repair efficiency indicates variant impact

Additional functional analyses include protein stability and expression assays, protein-protein interaction assays (particularly with MSH2), DNA binding assays, and ATPase activity assays. While in vitro assays have limitations including the artificial nature of experimental conditions, they provide valuable evidence that, when integrated with clinical, computational, and segregation data, enables more accurate classification of MSH6 variants.

What challenges exist in interpreting microsatellite instability (MSI) testing in MSH6 mutation carriers?

MSI testing in MSH6 mutation carriers presents several unique challenges that complicate interpretation:

• Lower Frequency of MSI-H Phenotype: 54% of colorectal and endometrial cancers from MSH6 mutation carriers showed no or only weak MSI in one study , creating potential for false negatives.

• Marker-Specific Patterns: MSH6-associated tumors often show instability predominantly at mononucleotide repeats rather than dinucleotide repeats , which may not be optimally detected by standard marker panels.

• Mutation-Type Variability: While truncating mutations more consistently lead to loss of protein expression and MSI, missense variants produce variable effects .

• Immunohistochemistry Discordance: Some tumors with loss of MSH6 staining may not show MSI-H, and conversely, some tumors with intact staining may exhibit MSI .

• Classification Limitations: The binary MSI-H/MSI-L classification may not adequately capture nuanced instability patterns in MSH6-deficient tumors.

What are the genotype-phenotype correlations observed with different types of MSH6 mutations?

Research has identified several important genotype-phenotype correlations in MSH6 mutation carriers:

These correlations reveal that despite significant differences in protein expression patterns between truncating and missense variants, their clinical manifestations are remarkably similar. This suggests that many missense variants significantly impair MMR function despite more subtle effects on protein expression, and supports considering them potentially pathogenic when assessing cancer risk .

How does MSH6 deficiency influence DNA repair pathway choice and genomic stability?

MSH6 deficiency impacts genomic stability through multiple mechanisms beyond simple failure of mismatch repair:

• Repair Pathway Interactions: MSH6 deficiency not only compromises MMR but affects other DNA repair pathways:

  • Base Excision Repair (BER): MSH6 recognizes certain oxidative DNA damage, and its deficiency may increase reliance on BER.

  • Homologous Recombination (HR): The MutSα complex (MSH2-MSH6) suppresses recombination between divergent sequences; MSH6 deficiency can lead to inappropriate recombination events.

• Mutation Spectrum: MSH6 deficiency produces a characteristic mutation signature:

  • Predominance of single-base substitutions over insertions/deletions

  • G:C → A:T transitions at non-CpG sites

  • Mutations clustered in regions of high replicative stress

• Cell Cycle Effects: The MSH6 protein has cell cycle-dependent expression and localization patterns that influence its repair function, with peak expression during S phase when DNA replication occurs.

• Tissue-Specific Consequences: The effects of MSH6 deficiency vary across tissues, which may explain the particular cancer spectrum seen in mutation carriers. Endometrial and colorectal tissues may be especially vulnerable to the types of DNA damage that go unrepaired in MSH6 deficiency.

Understanding these complex relationships is essential for developing targeted therapeutic approaches for tumors with MSH6 deficiency and for improving risk assessment in mutation carriers.