MUTYH the base excision repair gene family member associated with colorectal cancer polyposis.

COLORECTAL CANCER IS CLASSIFIED IN TO THREE FORMS: sporadic (70-75%), familial (20-25%) and hereditary (5-10%). hereditary colorectal cancer syndromes classified into two different subtypes: polyposis and non polyposis. Familial Adenomatous polyposis (FAP; OMIM #175100) is the most common polyposis syndrome, account for <1% of colorectal cancer incidence and characterized by germline mutations in the Adenomatous polyposis coli (APC, 5q21- q22; OMIM #175100). FAP is a dominant cancer predisposing syndrome which 20-25% cases are de novo. There is also another polyposis syndrome; MUTYH associated polyposis (MAP, OMIM 608456) which it is caused by mutation in human Mut Y homologue MUTYH (MUTYH; OMIM 604933) and it is associated with multiple (15-100) colonic adenomas. In this paper we discuss MUTYH mechanism as an important member of Base Excision Repair (BER) family and its important role in polyposis condition.

Introduction

It is estimated that 20,000 DNA damages occur in every cell per day (1). Gastrointestinal tract is a main target for oxidising elements which are highly mutationic (2). So colorectal cancer considered as a main cancer arises from exposure to this kind of agents. Beside Mismatch Repair (MMR) and Nucleotide excision repair (NER) Pathways which are the fundamental repair pathways interact with the mismatch pairs and aberrant nucleotide occurs in replication process, respectively, the base excision repair (BER) pathway is one of the main and primary DNA repair mechanisms that is involved in correcting the base mutations arised from oxidative, alkylation, deamination and depurination/depyrimidination damages (3). MUTYH is a DNA glycosylase and it belongs to BER family. The MUTYH protein is involved in the repair of post-replicative mispairs within DNA replication (4, 5).

MUTYH function and interactions

A human homologue of the Escherichia coli (E. coli) mutY gene was first cloned in 1996 (6), while the identification of the functional activity of the MUTYH gene first back in 2000 (7). This gene is called MUTYH and often known as hMYH or MYH, although this is not a correct name, because MYH is the gene symbol for the myosin heavy-chain gene. MUTYH is located on the short arm of chromosome 1(1p34.1) and spans 11.2 kb. This gene is a DNA glycosylase that is involved in the repair of post-replicative mispairs and plays a critical role in base excision repair (BER) pathway (4, 5). The oxidized form of guanine is 8-oxo-7, 8-dihydro-2’-deoxyguanosine (8-oxoG) which is considered as a frequent and stable element (8). In replication process 8-oxoG can pair with adenine as well as cytosine. The modified guanine (8-oxoG) is replicated in each round and the failure to remove the oxidized nucleotides before replication results in G: C to T: A transversion mutation (9, 10).

MUTYH mediates to remove A from A: 8-oxoG mispairs (7, 11) and OGG1 the other member of BER pathway detects and then removes 8-oxoG opposite cytosine (8-oxoG: C) (12, 13). Thus the cooperation of OGG1 and MUTYH together prevents G: C to T: A transversion mutation due to oxidative damages within replication process.

When an aberrant base is incised and then removed, it produce a gap called Apurinic/ A pyrimidinic (AP) sites which are mutagenic and should be corrected quickly (14). Completion of the repair process requires involvement of many additional proteins. More than 8 specific proteins detect specific DNA mutations which produce a basic or Apurinic/ A pyrimidinic (AP) sites (15).

Based on proteins involved the process of BER activity, there are two main mechanisms to repair AP site created by DNA damage: short patch repair pathway and long patch repair pathway (16).

Short-patch repair pathway involves making the association between POLB, APE1, XRCC1, PARP1, and either LIG1 or LIG3 genes. These related genes are activated when a single nucleotide insertion occurs and an AP endonuclease (also known as APE1 or APEX) incises the incorrect matched DNA at the AP site resulting in the formation of a 3́-hydroxyl end (3́OH) and a 5́ a basic sugar phosphate end (5́dRP) (17) Since MUTYH has no AP-lyase activity APE1 detects a basic site and then proceed the excision process. At the end, repair procedure of an aberrant nucleotide is accomplished by DNA ligase III (16).

A long-patch repair pathway requires PCNA, APE1, RFC, RPA, PARP1, FEN1, POLD/POLE and LIG1 for BER activity and they involve when 2–10 nucleotides mispaired in a DNA strand genes. In long patch repair pathway cleavage process accomplished by AP endonuclease (APEX1) and repair process is completed by proliferating cell nuclear antigen (PCNA) which has different types of functions include in DNA repair as well as cell cycle and DNA replication (3, 18).

Among repair genes, MUTYH is the main protein that detects peculiar A:G and A: 8-oxoG mispairs on DNA helix (19).

The MUTYH protein structure consists of many functional domains such as the N-terminal domain on the 5’ side and the C-terminal domain on the 3′ side. The N-terminal domain contains the catalytic region and includes a helix-hairpin-helix (HhH), pseudo HhH and an iron-sulfur cluster loop motif, which are also common region in other BER glycosylases; the C-terminal domain on the 3′ side of the MUTYH protein structure reported to have a role in recognition of 8-oxoG and shares homology with MTH1 (member of the BER family) (20–22).

Association between MUTYH and Replication Protein A (RPA), Proliferating Cell Nuclear Antigen (PCNA), p73, p53 and APE1has also reported in several studies (18, 23). Many papers suggested that in the damage condition, PCNA increases MUTYH activity (24, 25). Association of MUTYH gene and MMR genes such as MSH6, MSH2 and MLH1 has also been discussed (26–28). Although Most of the APC mutations produce truncated proteins, most pathogenic MUTYH variants are missense and splice site mutations and only a minority of variants are truncating mutations (29). The distribution of MUTYH mutations in MAP patients shows ethnic differences. Some variants are more common in other populations including: E480X in Indian (30), Y104X in Pakistani (31), c.1437_1439delGGA in Italian (32), c.1228_1229insGG in Portuguese (33), Q498H in German (34), and G25D and P18L in Chinese populations (35). Also, Y179C and G396D (previously known as Y165C and G382D) are two most common MUTYH mutations (80%) in Caucasian populations (30). Since only a few variants of MUTYH gene analyzed for their repair activity so far, it is recommended that other variants of MUTYH need to be examined for their involvement in pathogenesis of MAP (36).

Frequency of large deletions in MUTYH gene seem to be low and just Two papers revealed the presence of large deletions in MUTYH gene so far(5, 37). Loss of heterozygosity (LOH) of 1p is frequently happening in CRC tumors with chromosomal instability (CIN) (38). Since LOH is a common event in CRC tumors with CIN, LOH in MAP tumors display a distinct pattern of loss of heterozygosity with loss of parts of chromosomes without copy number alterations termed copy-neutral loss of heterozygosity which is not a frequent event in CRC tumors with CIN (39, 40). Croitoru et al showed that LOH detected in 20% of biallelic and 47% of monoallelic MUTYH mutation carriers (41). As demonstrated in several studies, microsatellite stable (MSS) is a dominant pattern of MSI in MAP tumors (42, 43).

MUTYH association polyposis characterizations

Mutation in MUTYH gene causes a predisposing condition to CRC termed MUTYH association polyposis (MAP) (2, 30). MAP was first reported by Al Tassan et al while they were evaluating ‘family N’. In this family three of seven siblings had a phenotype resemble with AFAP without aberrant mutation in APC gene, instead they observed that 11 tumors from three affected siblings had 18 somatic APC mutations which 15 mutations were G:C to T:A transversion mutations, this finding highlighted the possibility of deficiency in repair process of 8-oxoG mutations. They also reported that all three affecting siblings had biallelic mutation in MUTYH gene since it wasn't detected in rest of four siblings (2). Mean age at diagnosis of MAP patients is 48 and patients have between 10 and 100 colorectal polyps. The penetrance of this syndrome is 20–80% between 50 and 80 years (42–44). The phenotype of MAP patients resembles with Attenuated Familial Adenomatous polyposis coli AFAP (AFAP; OMIM #175100) individuals (30, 44).

Diagnosis of MAP patients with cases present overlapping features or AFAP patients is difficult since they share some similarities such as number of polyps, proximal location of polyps and early onset of CRC (42–45). In MAP patients Polyps are frequently small and mostly located left-side of the colon (42). Proximal location of polyps is the key point to distinguish cases with moderate adenomatous polyps from those of sporadic (42, 46). In comparison with the general population CRC risk in MAP patients, associated with 28- to 93-fold (42, 47). Histopathologically Adenomas (tubular or tubulo-villous) are detected in entire colon consider as predominant lesions in AFAP/FAP and also in MAP patients. Since serrated polyps: hyperplastic polyps, sessile serrated polyps (also referred to as sessile serrated adenomas) and traditional serrated adenomas are not present in Affected harbouring mutation in APC gene, they are common types of lesions in MAP patients (48). Finally APC genetic testing for this group of patients with serrated polyps wouldn't be informative. Tumors in MAP patients show a high frequency of distinctive somatic G:C to T:A mutations in the APC and Kras genes (2, 30). GAA sequences in APC gene are the target sites for truncating mutations and this site is frequently mutated during tumorigenesis (2, 30, 49). APC has 216 GAA sites in which G:C→T:A mutations could happen and result in a termination codons (2). In contrast TP53, PTCH, RB1, NF1 and VHL have fewer target sites and this makes the APC the best target than the other genes for mutagenesis in MAP (tumors (49). It is notable that, in Kras gene the hot spot codon is c.34G > T at codon12 (50, 51).

Screening and Management

Early detection, genetic counseling and MUTYH mutation screening are important in affected individuals and their siblings. Based on National Comprehensive Cancer Network NCCN recommendation, Colonoscopy starts at age 25 years (52) and patients with more than 10 adenomas should be referred for genetic counseling and testing procedure (52). Patients with less than 10 adenomas should be referred for follow up screening and genetic testing for this group of patients is not necessary.

Other surveillance protocol for MAP patients recommends similar screening program similar AFAP patients. Patients undergo colonoscopy every 2 years starting at 18–20 years and upper gastrointestinal endoscopy starts when affected is between 25 and 30 years of age (53, 54).

MUTYH mutation screening is recommended for people who are diagnosed with MAP and patients who have a recessive mutation transmission and phenotype similar to AFAP. The affected may not be seen in every generation and usually have a normal parents (55, 56).

First of all, the two putative codons Y165C or G382D are examined for their high incidence rate in majority of populations. Then PCR sequencing is performed for the entire coding region and intron–exon boundaries of MUTYH. The genetic testing for MUTYH in patients with multiple serrated polyps without adenomas is not recommended by NCCN (52).

There haven't been any reports to define molecular screening in patients with MAP in Iran yet and Research is ongoing to determine APC and MUTYH variants in FAP patients. But screening of mutations in other Genes associated with CRC carried out and other repair genes like MLH1 and MSH6 have been studied (57–60).

Prevalence

Approximately 0.3%–1% of all colorectal cancers is associated with MAP (41, 42).

It is estimated that 1% to 2% of the general population has a mutation in MUTYH. There isn't a peculiar criterion to classify nonpolyposis MUTYH-associated CRC phenotype, so it has been recommended that all early-onset CRC cases should be evaluated for MUTYH mutations (61). Several Studies demonstrated that up to 30% of biallelic MUTYH mutation carriers develop CRC although they do not present a polyposis condition (62). There has also been reported some cases with MAP and no polyps whereas in some cases more than 500 colorectal polyps observed (44). APC germline mutations are not present in 10–30% of FAP patients and in up to 90% of AFAP patients (63). In another word, APC mutations are detected in 10–22% of AFAP cases and biallelic germline mutation of MUTYH were identified in 15–30% of AFAP patients and approximately 7–22% of FAP patients. Biallelic MUTYH mutations can be detected in 30% of APC mutation-negative patients. (30, 55, 64, 65).

Biallelic germline mutations in MUTYH are common in patients with negative APC related FAP patients and in MAP cases but recently studies are focused on monoallelic MUTYH variants in CRC patients and try to identify the association between monoallelic mutation susceptibility to CRC (66–68).

Monoallelic MUTYH mutation carrier's account for 1% to 2% of the general population since Biallelic mutations observed in less than 1% of all CRCs and the frequency of this mutation in patients with10 to 100 polyps are 28% and in individuals with 100 to 1000 polyps are 14% (44, 69).

First degree relatives of MAP patients with biallelic mutation in MUTYH gene are considered as obligate carriers who carry at least one MUTYH mutation. Both parents are carriers of a biallelic mutation and each child has 25% chance of inheriting two mutations. Whether monoallelic MUTYH carriers (heterozygote) are at high risk for developing CRC is still not clear: compared with the general population There is evidence that obligate monoallelic MUTYH mutation carriers have a modest risk for colorectal cancer (47, 66). Some authors believe that heterozygote mutation carriers should consider as low penetrance alleles, although consensus surveillance guidelines for this subgroup need to be developed.

MUTYH and other cancers

Extracolonic manifestations are common in patients with MAP which include: duodenal cancer and related polyposis, cancers such as gastric, small intestinal, endometrial, liver, ovarian, bladder, thyroid, Breast and skin cancers including melanoma, squamous epithelial, and basal cell carcinomas (70, 71). Other manifestations such as osteomas, dental cysts and congenital hypertrophy of the retinal pigment epithelium (CHRPE) are also seen in this group of patients (32, 71, 72). These extra colonic manifestations are also reported in FAP patients and the occurrence is less in MAP than in FAP or AFAP patients (73). The association between breast cancer and MUTYH gene is not defined clearly so far (71). Since Frequency of biallelic MUTYH mutations in breast cancer seem to be low (74, 75) in valuable paper by Wasielewski et al. reported heterozygote mutations in MUTYH gene in families with both CRC and breast cancer moreover they have also reported that there was an increased risk for breast cancer in Female MAP patients (76). The association of many malignancy such as endometrial (77, 78), Gastric(79, 80), Bladder(81) lung(82) and Diabetes(83) with MAP have reported respectively. Screening of Somatic MUTYH gene Mutations in sporadic CRC patients doesn't seem to be informative since the majority of papers revealed no association between MUTYH and sporadic colorectal cancer (84, 85) except one paper detected two MUTYH mutations in Tunisian patients (86).

Immune system response

The immune system of patients with deficiency in DNA mismatch repair genes is more active than individuals without DNA repair defects. It is proposed that the accumulation of peptide elements and mutant proteins in surface of the tumor cells in patients with high MSI or aberrant expression of MMR genes, makes the immune system more active then this results in better diagnosis and survival (87, 88). This active immune system could affect tumorigenesis while Antigens presenting in the tumor cell surface (89). Infact The accumulation of peptides and neoantigenes in such patients simulate anti-tumor immune response (90) and in comparison with the other lesions they show increased survival rates (91). High Infiltration of lymphocytes in MAP tumors reported in several studies previously (43, 51). Human leukocyte antigen class I complexes mediates in targeting of tumor cells by CD8+ and loss of expression of this antigen is a common event in MSI-H and MAP tumors (92–94). When HLA is lost then tumors hide from immune system due to deficiency in recognition and elimination (95, 96).

Conclusion

Although the MUTYH mutations gaining attention in diagnosis and counseling of patients with CRC and polyposis, many questions about diagnostic and screening protocols still remained unanswered. Detection of MUTYH gene variants and their association with polyposis and non polyposis CRC and study of immune system molecules and their involvements in tumorigenesis of MAP patients will be worthwhile for better diagnosis and further screening schedule for MAP patients.