Microsatellite instability (MSI) testing and Methylation assay Practical
Background information
There are two major genetic pathways leading to colorectal cancer. About 80% of colon cancers are caused by chromosomal instability, with gross changes in chromosome number and integrity. These are microsatellite stable (MSS). Microsatellite instability (MSI) is observed in about 10-15% of sporadic colon carcinomas. The vast majority of colon cancers occur sporadically in older individuals, but a few have family histories.
Microsatellite Instability (MSI)
Microsatellites are short repeated DNA sequences, consisting of mono-, di-, tri- and tetra-nucleotides in tandem, and are found throughout the genome. Microsatellite repeats show length polymorphisms, due to variable numbers of the repeating unit in different individuals. Because they are repetitive, microsatellites are prone to strand slippage and replication errors.
MSI – a DNA aberration characterized by altered repeat numbers in microsatellite repeat sequences in cancer cells compared to normal cells from the same individual. Deletions or insertions within the repeats lead to an altered allelic size in cancer cells displaying the MSI phenotype.
MSI and DNA mismatch repair
MSI is caused by defects in DNA mismatch repair enzymes encoded by the genes MLH1, MSH2, MSH3, PMS1, PMS2, MLH3, and MSH6. These enzymes normally proof-read and correct nucleotide base-pair mistakes made during DNA replication. Impaired DNA mismatch repair activity leads to the accumulation of mutations in other genes, and MSI.
The detection of MSI is a useful molecular screening tool for cancer with defects in DNA mismatch repair. This provides information regarding the cause of cancer and aids in choosing diagnostic and chemotherapeutic modalities.
Genes with simple repeats in their coding regions are susceptible to deletions in MSI cancers. Several genes implicated in tumourigenesis have repeats in the coding regions and are commonly somatically inactivated by frameshifts causing deletions e.g., TGFβRII, BAX, hMSH3, IGFIIR and hMSH6.
Loss of DNA mismatch repair activity:
There are three main mechanisms leading to loss of a functional DNA mismatch repair gene:
1)Mutation,
2)Loss of heterozygosity
3)“Epigenetic” inactivation due to methylation of the gene promoter
Both alleles of the gene are lost before cancer starts, referred to as “Knudsen’s two-hit hypothesis”. Familial cases have inherited mutations, often followed by somatic loss of heterozygosity of the second allele. Most sporadic cases have methylation of the promoter.
Hereditary non-polyposis colorectal cancer (HNPCC)
MSI cancers were first described in the context of HNPCC.
From kindred’s with HNPCC, MSI is detected in as many as 85-95% of cases. The key characteristic of HNPCC is MSI caused by a defective DNA mismatch repair system. HNPCC accounts for about 3-5% of all colorectal cancer.
For patients with suspected HNPCC, an attractive, cost-effective strategy is to first perform MSI testing on the affected family member’s colorectal tumour and if the tumour is found to exhibit MSI, then the patient/family may consider germline testing for mutations in the hMSH2 and hMLH1 genes, which in combination account for 60% of HNPCC cases.
MSI +ve pathways proceed with little evidence of loss-of-heterozygosity (LOH), whereas MSS (MSI-negative) cancers show a loss of classical tumour suppressor loci. In contrast to MSI where new alleles are generated, LOH is correlated with loss of wild type allele in tumor DNA.
Sporadic colon cancers
The majority of sporadic MSI cancers arise by MLH1 silencing via an epigenetic mechanism (methylation of the promoter) that inactivates both MLH1 alleles. Methylation of MLH1 is thus said to be “biallelic”.
Loss of Heterozygosity (LOH)
Loss of heterozygositystudies map regions of allelic loss using microsatellite markers.
LOH of polymorphic markers in tumour compared with normal tissue is a sign of somatic deletion.
Mechanisms of LOH:
Deletion of
the normal allele
the chromosome arm containing the normal allele
the entire chromosome containing the normal allele (resulting in aneuploidy).
LOH can only be assayed at heterozygous loci and paired normal and tumour DNA from an individual must be available.
Quantitation of LOH
LOH is calculated from the area under the curve (with reference to peak height) using a ratio of alleles calculation:
Allele 1-normal/Allele 2-normal
Allele 1-tumour/Allele 2-tumour
LOH is positive if X<0.5 or >2.0
MSI Markers
The designation of a colon cancer as showing MSI depends on the detection of at least 2 unstable loci out of 5, from a panel of loci that were selected during a National Cancer Institute consensus conference. Of the 5 microsatellites tested, three are mononucleotide and two are dinucleotides.
By consensus, microsatellite status has been divided into 3 groups
1)microsatellite stable (MSS) - no instability seen
2)low-level instability (MSI-L) – 1 locus unstable
3)high-level instability (MSI-H) – 2 or more loci unstable
MSI-L tumours appear and behave more like MSS cancers
BATS- Mononucleotide repeats
BAT-25
Intragenic to the c-kit protooncogene
a locus with a span of 25 adenosines (i.e., poly (A))
considered monomorphic (quasi-monomorphic) with allelic size variation not exceeding two nucleotides; little need to compare the allelic profiles between N and T.
BAT-26
located in an intron of hMSH2
a locus with a span of 26 adenosines that varies little within the population.
monomorphic-probably the best mononucleotide to establish status without matching normal DNA
BAT-40
located in an intron of the 3--hydroxysteroid dehydrogenase gene
polymorphic
Due to the extremely high homozygosity of mononucleotide repeats in the general population, BAT 25 and BAT 26 are more sensitive and better markers for microsatellite instability detection than their dinucleotide counterparts.
Dinucleotide repeats
D5S346
located on chromosome 5
a (CA)n repeat marker located within a gene called DP-1.
utilised as a microsatellite marker for the APC gene
D17S250
a (CA)n repeat marker located on chromosome 17
Laboratory Methodology
Amplified fluorescent PCR products are analyzed using an automated ABI sequencer. The products are electrophoresed on a gel to separate the PCR fragments (dependent on number of repeat units), then the fluorescence is detected by the lazer and recorded.
MSI is defined by the presence of novel peaks, following the amplification of tumour DNA that was not present in normal DNA.
A typical phenomenon of MSI amplification is the generation of a main band and several peaks (stutter bands) per allele during PCR amplification as a consequence of polymerase slippage.
Methylation
Methylation in mammals occurs in the cytosine base at CG dinucleotides only. Gene promoters have dense levels of CGs, but in order to keep the gene active, these normally remain unmethylated. Methylation of most CGs in the promoter causes the genes to be silenced. Methylation causes the gene to be shut-off by preventing binding of the transcriptional machinery to the gene promoter, which normally keeps it running. The accumulation of methylation at gene promoters is age-related.
Assay for Methylation of genes
Combined bisulphite and restriction analysis “COBRA”.
Treatment of DNA with sodium bisulphite (NaHSO3) causes unmethylated cytosines to become uracils, whereas methylated Cs are unreactive and so stay as a C. The C/T differences in the treated DNA allow methylated DNA to be discriminated from unmethylated DNA using chosen restriction enzymes that recognize and cut one sequence but not the other. The DNA is then electrophoresed on an agarose gel to separate the fragments on the basis of size, so that cut and uncut bands can be detected as positive or negative for methylation.
C U T TTGA (no enzyme cut site)
NaHSO3 PCR Digestion
C5m C C TC/GA (TaqI enzyme cut site)
“COBRA” assay for the detection of methylation at the MLH1 promoter.
Forward PCR primer Restriction enzyme “TaqI”
1201 aagttCGgtt tCGgtatttttgtttttatt ggttggatat ttCGtatttt tCGagttttt
1261 aaaaaCGaat taataggaag agCGgatagC GatttttaaC GCGtaagCGt atattttttt
1321 aggtagCGgg tagtagtCGt tttagggagg gaCGaagaga tttagtaatt tatagagttg
1381 ag Reverse PCR primer
PCR product size = 170 bp
If methylated, TaqI will cut the PCR product to form two fragments of 31bp and 139bp. If unmethylated, the enzyme fails to cut and the original band remains at 170bp.
Practical class
Please form pairs to work in.
Each pair will be asked to determine whether there is methylation of the MLH1 promoter (as above) by performing a restriction digest of ready-made PCR products for patients with case history 1 and 2. The digests will be loaded on an agarose gel and electrophoresed to obtain the result. In addition you will be given some graphical readouts from MSI and LOH tests performed for patients 1 and 2, and you will be asked to interpret the data. Please bring a lab coat and these sheets to help you with the data interpretation.NB No eating or drinking is permitted in the lab.