MLH1 Mutations and the Formation of Hereditary Non-Polyposis Colon Cancer(Lynch Syndrome) Kevin Harris Hereditary Non-polyposis Colorectal Cancer (HNPCC) is an inherited colorectal cancer syndrome accounting for approximately 5 percent of all cases of colorectal cancer. This disease is characterized by the formation of colon cancer with very few or no observable polyps present. Tumors often form in the patient’s ascending colon. Microsatellite instability observed in tumor biopsies is often an indication HNPCC facilitated the tumor formation.

Some Families are Predisposed to Colon Cancer Physicians have been studying HNPCC for over 100 years. Doctors realized certain families were predisposed to develop colon cancer. By creating family pedigrees scientists made some interesting findings. First they noted men and women were roughly equally affected by HNPCC. Second they noted approximately 50% of the offspring of an affected individual are also affected by HNPCC. This led them to the conclusion HNPCC is inherited in an autosomal dominant fashion. Some Families are Predisposed to Colon Cancer

Scientists mapped the genes responsible for causing HNPCC and confirmed their hypothesis that is was an autosomal dominate disorder. Many genes are responsible for causing HNPCC. The MSH6, MSH2 and PSM1 genes are located on chromosome 2. The PSM2 gene is located on chromosome 7. The MLH1 gene which encodes MLH1 protein is located on chromosome 3. It is important to know which gene is responsible for causing HNPCC in a family because physicians will often personalize treatment plans based on this information. Furthermore, patients with different types of mutations carry different risk profiles. We focus on MLH1 mutations because they are responsible for approximately 50% of HNPCC mutations. Sequence variants such as point mutations account for 90-95% of MLH1 mutations. What Genes Cause HNPCC?

MLH1 Encodes a DNA Mismatch Repair Protein MLH1 encodes a DNA mismatch repair protein. The DNA mismatch repair complex is highly conserved between eukaryotes and prokaryotes. Most of the initial research of this complex occurred in E. Coli in the early 1990s. MLH1 is the human homologue for the E. Coli DNA mismatch repair gene MutL. MLH1 stands for MutL homologue 1. In humans the DNA mismatch repair (MMR) machinery acts by a three step process of licensing, degradation and resynthesis. MutS alpha or MutS beta bind mismatched DNA and recruit MLH1 to the area. The MMR machinery is largely refractory to covalent double stranded DNA and therefore a nick must be present in order for the MutS alpha or MutS beta subunits to bind. MLH1 is already bound to PMS2. When MLH1 binds to MSH2 is causes the complex to diffuse along the DNA until it encounters PCNA. EXO1 (Exonuclease 1) removes nucleotides and creates a single stranded gap in DNA. EXO1 has a obligate 5’ to 3’ directionality but PMS2 has a cryptic endonuclease activity. If the match repair machinery needs to remove nucleotides on the 3’ side of a nick PMS2 is able to introduce additional single stranded breaks into the pre-nicked strand. Following the removal of all mismatched nucleotides DNA is resynthesized by Polymerase delta and nicks are fixed by DNA ligase. MLH1 Encodes a DNA Mismatch Repair Protein

Meiotic Pachytene Arrest is Observed in MLH1-Deficient Mice To understand the role of MLH1 in normal growth and development, scientists generated mice that have a null mutation of this gene. Mice homozygous for this mutation show a replication error phenotype, and extracts of these cells are deficient in mismatch repair activity. Homozygous mutant males show normal mating behavior but have no detectable mature sperm. Examination of meiosis in these males reveals the cells enter meiotic prophase and arrest at pachytene. Homozygous mutant females have normal estrous cycles and reproductive and mating behavior but are infertile. The phenotypes of the mlh1 mutant mice are distinct from those deficient in msh2 and pms2. The different phenotypes of the three types of mutant mice suggest that these three genes may have independent functions in mammalian meiosis. Figure A shows the knockout scheme performed by homologous recombination in mice. Figure B is a southern blot confirming the proper genotype in knockout mice. The fact that homozygous mutants (-/-) are viable suggests that MLH1 is not essential for embryonic development. Meiotic Pachytene Arrest is Observed in MLH1-Deficient Mice

MLH1 Mutants Lack MLH1 Protein and Make Many Replication Errors Detection of MLH1 Protein by Western Blot Analysis. H, is MLH1 from human lymphoblastoid cell line. It is evident that no 90 kD band is present in the homozygous mutant. MLH1 Mutants Lack MLH1 Protein and Make Many Replication Errors

Molecular Markers Confirm MLH1 Mutations in HNPCC Patients

Familial Non-Polyposis Colon Cancer (Lynch Syndrome) Leads to Other Cancers

Polyps Come in Many Forms FAP HNPCC Polyps Come in Many Forms

With HNPCC Additional Screening Visits are Necessary

A Preemptive Strike Against Cancer Currently there are not many chemotherapeutic agents available to patients with HNPCC. Many HNPCC patients chose to under go prophylactic surgery to remove their colon before cancer forms. Genetic counselors are able to educate their patients about the benefits and risks associated with prophylactic surgeries. A recent study completed in 2010 showed a 63% decrease in the likelihood of developing colon cancer if patients took 600 mg of Aspirin per day. Researchers were very pleased with these results and believe there could be implications that extend beyond Lynch syndrome. According to one scientist, “There is no reason to believe the cancers that develop in Lynch syndrome are any different biologically that those in the general population”. A Preemptive Strike Against Cancer

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