DNA MISMATCH REPAIR PROTEIN MLH3; MLH3

Alternative titles; symbols

• MutL, E. COLI, HOMOLOG OF, 3

HGNC Approved Gene Symbol: MLH3

Cytogenetic location: 14q24.3 Genomic coordinates (GRCh38): 14:75,013,763-75,051,478 (from NCBI)

▼ Cloning and Expression
DNA mismatch repair (MMR) is important because of its role in maintaining genomic integrity and its association with hereditary nonpolyposis colon cancer (HNPCC; see 120435). To identify new human mismatch repair proteins, Lipkin et al. (2000) probed nuclear extracts with the conserved C-terminal interaction domain of MLH1 (120436). They described the cloning and complete genomic sequence of MLH3, which encodes a DNA mismatch repair protein that interacts with MLH1. They found that MLH3 is more similar to mismatch repair proteins from yeast, plants, worms, and bacteria than to any known mammalian protein, suggesting that its conserved sequence may confer unique functions in mice and humans. Cells in culture stably expressing a dominant-negative MLH3 protein exhibited microsatellite instability.

▼ Gene Function
To investigate whether MLH3 acts during meiotic recombination, (Santucci-Darmanin et al. (2002)) analyzed its expression in mammalian germ cells. The MLH3 gene was expressed in mouse meiotic cells and in human testis, and immunoprecipitation assays revealed that the MLH3 protein was found in mouse spermatocytes. The meiosis-specific MSH4 (602105) protein, known to participate in meiotic recombination, coimmunoprecipitated with MLH3 from mouse meiotic cell extracts. Two MLH3 protein isoforms potentially expressed in human testis (MLH3 and MLH3-delta-7) interacted in vitro with the MSH4 protein. The authors suggested that MLH3 is associated with MSH4 in mammalian meiotic cells, and that MLH3 may play a role in mammalian meiotic recombination.

Cannavo et al. (2020) showed that human MutS-gamma, a complex of MSH4 and MSH5 (603382) that supports crossing over, bound branched recombination intermediates and associated with MutL-gamma, a complex of MLH1 (120436) and MLH3, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutS-gamma directly stimulated DNA cleavage by the MutL-gamma endonuclease. MutL-gamma activity was further stimulated by exonuclease-1 (EXO1; 606063), but only when MutS-gamma was present. Replication factor C (RFC; see 102579) and proliferating cell nuclear antigen (PCNA; 176740) were additional components of the nuclease ensemble, thereby triggering crossing over. S. cerevisiae strains in which MutL-gamma could not interact with Pcna presented defects in forming crossovers. The MutL-gamma-MutS-gamma-EXO1-RFC-PCNA nuclease ensemble preferentially cleaved DNA with Holliday junctions, but it showed no canonical resolvase activity. Instead, the data suggested that the nuclease ensemble processed meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points. The authors proposed that, since DNA nicking by MutL-gamma depends on its cofactors, the asymmetric distribution of MutS-gamma and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. They suggested that this mode of MutL-gamma nuclease activation may explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes.

Independently, Kulkarni et al. (2020) showed that PCNA was important for crossover-biased resolution. In vitro assays with human enzymes showed that PCNA and RFC were sufficient to activate the MutL-gamma endonuclease. MutL-gamma was further stimulated by the codependent activity of the pro-crossover factors EXO1 and MutS-gamma, the latter of which binds Holliday junctions. The authors found that MutL-gamma also bound various branched DNAs, including Holliday junctions, but it did not show canonical resolvase activity, suggesting that the endonuclease incises adjacent to junction branch points to achieve resolution. In vivo, Rfc facilitated MutL-gamma-dependent crossing over in budding yeast. Moreover, Pcna localized to prospective crossover sites along synapsed chromosomes. Kulkarni et al. (2020) concluded that their data highlight similarities between crossover resolution and the initiation steps of DNA mismatch repair and evoke a novel model for crossover-specific resolution of double Holliday junctions during meiosis.

▼ Mapping
By fluorescence in situ hybridization using mouse and human BACs, Lipkin et al. (2000) mapped the respective MLH3 genes to human 14q24.3 and mouse 12.

▼ Molecular Genetics
Somatic Mutation in Colorectal Cancer

Malfunction of the mismatch repair system results in a mutator phenotype, which is manifested as microsatellite instability (MSI). MSI is often divided into 2 forms: MSI-high (MSI-H) and MSI-low (MSI-L), based quantitatively on the observed frequency of genomic mutations (Boland et al., 1998). Lipkin et al. (2001) screened 36 colon tumors and discovered an appreciable frequency of somatic MLH3 coding mutations in MSI-H tumors (25%). They found mutations in 8- and 9-bp polyadenine mononucleotide runs that resulted in frameshift. The 8-bp run extended from coding nucleotides 1747 to 1755; the 9-bp run extended from 2014 to 2021. In 4 of 6 tumors, evidence of biallelic inactivation was noted. Furthermore, MLH3 nonsense mutations were identified in 2 of 12 microsatellite-stable (MSS) tumors with 14q24 loss of heterozygosity (see 604395.0001). Screening of 60 probands with increased genetic risk factors for colorectal cancer susceptibility demonstrated no germline mutations of MLH3 and no mutations in other candidate genes. While the analyses did not exclude the existence of germline MLH3 mutations in some such patients, they suggested that they are at most uncommon. The finding of an appreciable frequency of somatic MLH3 mutations was considered consistent with a possible role for this gene in the progression of colorectal cancer tumorigenesis.

Hereditary Nonpolyposis Colorectal Cancer 7

Wu et al. (2001) investigated the possible role of MLH3 in hereditary nonpolyposis colorectal cancer by scanning for mutations in 39 HNPCC families and in 288 patients suspected of having HNPCC. They identified 10 different germline MLH3 variants, 1 frameshift and 9 missense mutations, in 12 patients suspected of HNPCC. In 3 of the 12 patients, a mutation was also found in MSH6 (600678). Eight of the 10 mutations were situated in exon 1, 1 was in exon 11, and 1 was in exon 12. The same group (Ou et al., 2009) found that all of the reported MLH3 variants were expressed normally, localized normally in the cell nucleus, and interacted normally with MLH1. Ou et al. (2009) concluded that there is no evidence to support a role for MLH3 variants in HNPCC, although a role for such variants in tumorigenesis cannot be fully excluded.

Liu et al. (2003) identified 12 variants in the MLH3 gene (see, e.g., 604395.0005-604395.0008) in 16 (23%) of 70 probands of families with colorectal cancer (HNPCC7; 614385), some of whom had relatives with endometrial cancer. Most mutations showed reduced penetrance, suggesting that MLH3 is a low-risk gene and may work together with other factors in an additive manner. None of the tumors with MLH3 mutations showed microsatellite instability, indicating that MLH3 does not contribute to carcinogenesis through impaired DNA mismatch repair function.

Esophageal Cancer

In a cohort of patients with esophageal cancer (133239), Liu et al. (2006) found that while MLH3 is a high-risk gene with a reduced penetrance in some families, it acts as a low-risk gene in most families, and may work together with other genes in an accumulated manner. They concluded that MLH3 mutations may predispose to esophageal cancer in some families.

Endometrial Cancer

Taylor et al. (2006) analyzed the MLH3 gene in 57 women with endometrial cancer (608089). One patient had a germline variant (T942I) and loss of heterozygosity at the MLH3 locus in the tumor tissue. A germline heterozygous P844L polymorphism was found in 61% of patients. Somatic MLH3 mutations were identified in 3 of 57 tumors. Functional expression studies were not performed. Taylor et al. (2006) concluded that MLH3 mutations may play a role in a subset of endometrial cancers.

▼ Animal Model
Lipkin et al. (2000) stated that mouse Mlh3 is highly expressed in gastrointestinal epithelium and physically maps to the mouse complex trait locus colon cancer susceptibility-1 (Ccs1). Although Lipkin et al. (2000) were unable to identify a mutation in the protein-coding region of Mlh3 in the susceptible mouse strain, colon tumors from congenic Ccs1 mice exhibited microsatellite instability. Functional redundancy among Mlh3, Pms1 (600258), and Pms2 (600259) may explain why neither Pms1 nor Pms2 mutant mice develop colon cancer, and why PMS1 and PMS2 mutations are only rarely found in HNPCC families.

To assess the role of Mlh3 in mammalian meiosis, Lipkin et al. (2002) generated and characterized Mlh3 -/- mice. They showed that the null mice are viable but sterile. Mlh3 is required for Mlh1 binding to meiotic chromosomes and localizes to meiotic chromosomes from the mid-pachynema stage of prophase I. Mlh3 -/- spermatocytes reached metaphase before succumbing to apoptosis, but oocytes failed to complete meiosis I after fertilization. The results showed that Mlh3 has an essential and distinct role in mammalian meiosis.

▼ ALLELIC VARIANTS ( 8 Selected Examples):

.0001 COLORECTAL CANCER, SOMATIC
MLH3, 2483G-T
In a colorectal cancer (114500) sample that showed loss of heterozygosity at 14q24, Lipkin et al. (2001) identified a 2483G-T transversion that converted codon GAG (glu) to TAG (stop) in the MLH3 gene.

.0002 RECLASSIFIED - VARIANT OF UNKNOWN SIGNIFICANCE
MLH3, GLN24GLU
This variant, formerly titled COLON CANCER, HEREDITARY NONPOLYPOSIS, TYPE 7, has been reclassified based on the findings of Korhonen et al. (2008) and Ou et al. (2009).

Wu et al. (2001) identified a 70C-G transversion in exon 1 of the MLH3 gene, resulting in a missense gln24-to-glu (Q24E) amino acid change in a patient with HNPCC. There was no associated mutation found in MSH6 (600678).

By in vitro functional expression studies, Ou et al. (2009) determined that the Q24E MLH3 variant was expressed normally, localized normally in the cell nucleus, and interacted normally with MLH1 (120436). In silico analysis suggested no damaging effect of the change. Independent studies by Korhonen et al. (2008) showed that the Q24E variant functioned normally and was able to complement mismatch repair defects in cell lines. Ou et al. (2009) concluded that there is no evidence to support a role for this variant in HNPCC, although a role for the variant in tumorigenesis cannot be fully excluded.

.0003 RECLASSIFIED - VARIANT OF UNKNOWN SIGNIFICANCE
MLH3, ASN499SER
This variant, formerly titled COLON CANCER, HEREDITARY NONPOLYPOSIS, TYPE 7, has been reclassified based on the findings of Ou et al. (2009).

Wu et al. (2001) identified a 1496A-G transition in exon 1 of the MLH3 gene, resulting in an asn499-to-ser (N499S) amino acid change, in a patient with HNPCC.

By in vitro functional expression studies, Ou et al. (2009) determined that the N499S MLH3 variant was expressed normally, localized normally in the cell nucleus, and interacted normally with MLH1 (120436). In silico analysis suggested a possibly damaging effect of the change. Ou et al. (2009) concluded that there is no evidence to support a role for this variant in HNPCC, although a role for the variant in tumorigenesis cannot be fully excluded.

.0004 RECLASSIFIED - VARIANT OF UNKNOWN SIGNIFICANCE
MLH3, GLU624GLN
This variant, formerly titled COLON CANCER, HEREDITARY NONPOLYPOSIS, TYPE 7, has been reclassified based on the findings of Ou et al. (2009).

In 2 unrelated probands with HNPCC, Wu et al. (2001) found an 1870G-C transversion in exon 1 of the MLH3 gene, predicted to result in a glu624-to-gln (E624Q) amino acid change. Immunohistochemical analysis demonstrated the expression of MSH2 (609309), MLH1 (see 120436), and MSH6 (600678) in 1 patient.

Liu et al. (2003) identified the E624Q substitution in 1 patient with familial colorectal cancer. However, none of 6 other affected family members carried this variant, and it was found in 3.2% of controls.

By in vitro functional expression studies, Ou et al. (2009) determined that the N499S MLH3 variant was expressed normally, localized normally in the cell nucleus, and interacted normally with MLH1 (120436). In silico analysis suggested no damaging effect of the change. Ou et al. (2009) concluded that there is no evidence to support a role for this variant in HNPCC, although a role for the variant in tumorigenesis cannot be fully excluded.

.0005 RECLASSIFIED - VARIANT OF UNKNOWN SIGNIFICANCE
MLH3, GLU1451LYS
This variant, formerly titled COLON CANCER, HEREDITARY NONPOLYPOSIS, TYPE 7, has been reclassified based on the findings of Ou et al. (2009) and Korhonen et al. (2008).

In 2 probands with HNPCC, Wu et al. (2001) identified a heterozygous a 4351G-A transition in exon 12 of the MLH3 gene, predicted to result in a glu1451-to-lys (E1451K) amino acid substitution. Both probands were also compound heterozygous for a mutation in the MSH6 gene (V878A; 600678.0006 and 650insT; 600678.0007, respectively).

Liu et al. (2003) identified the E1451K mutation in a patient with colorectal cancer and in her sister with endometrial cancer (608089). However, the mutation was not found in another sister with colorectal cancer and was not found in 90 control individuals.

Kim et al. (2007) identified the E1451K variant in healthy Korean controls and concluded that it is a polymorphism in that population.

By in vitro functional expression studies, Ou et al. (2009) determined that the E1451K MLH3 variant was expressed normally, localized normally in the cell nucleus, and interacted normally with MLH1 (120436). In silico analysis suggested no damaging effect of the change. Independent studies by Korhonen et al. (2008) showed that the E1451K variant functioned normally and was able to complement mismatch repair defects in cell lines. Ou et al. (2009) concluded that there is no evidence to support a role for this variant in HNPCC, although a role for the variant in tumorigenesis cannot be fully excluded.

.0006 COLORECTAL CANCER, HEREDITARY NONPOLYPOSIS, TYPE 7
ENDOMETRIAL CANCER, INCLUDED
MLH3, 1-BP DEL, 885G
In a patient with colorectal cancer (HNPCC7; 614385), Liu et al. (2003) identified a 1-bp deletion (885delG) in exon 1 of the MLH3 gene, predicted to result in a frameshift and premature termination. The mutation was found in another family member with colorectal cancer, in 1 family member with endometrial cancer (608089), and in 1 of 3 unaffected relatives over the age of 75 years, indicating reduced penetrance. The mutation was not found in 96 controls.

.0007 ENDOMETRIAL CANCER
COLORECTAL CANCER, HEREDITARY NONPOLYPOSIS, TYPE 7, INCLUDED
MLH3, VAL741PHE
In a mother and daughter with endometrial cancer (608089), Liu et al. (2003) identified a heterozygous 2221G-T transversion in exon 1 of the MLH3 gene, resulting in a val741-to-phe (V741F) substitution. An unaffected aunt, over the age of 80 years, also carried the mutation, indicating reduced penetrance. The mutation was not found in 95 controls.

Kim et al. (2007) identified a V741F mutation in a 71-year-old man with colon cancer (HNPCC7; 614385). His 2 sisters developed gastric cancer and breast cancer at ages 57 and 61, respectively. The authors suggested moderate penetrance for this variant.

.0008 COLORECTAL CANCER, HEREDITARY NONPOLYPOSIS, TYPE 7
MLH3, TRP1276ARG
In 4 sibs with HNPCC (HNPCC7; 614385) without microsatellite instability, Liu et al. (2003) identified a 3826T-C transition in exon 7 of the MLH3 gene, resulting in a trp1276-to-arg (W1276R) substitution. The mutation was inherited from the mother, who had gastric cancer. The mutation was not found in 96 controls. All 4 sibs also carried a mutation in the MSH2 gene (609309). Liu et al. (2003) suggested that the additive effect of these 2 mutations resulted in the phenotype. These findings were consistent with the hypothesis that low-penetrance additive risk alleles contribute to the risk of developing colorectal cancer.

Liu et al. (2006) identified a W1276R substitution in 1 patient with colorectal cancer. However, 2 unaffected family members also carried the variant and 2 family members with esophageal cancer did not carry the mutation.