Clinical correlation:

Presently, molecular techniques are used in the detection of Factor V Leiden gene mutation.

Factor V is an important protein that acts in the coagulation cascade. Mutation of this gene results in increased risk of thromboembolism (sometimes, termed hypercoagulability). Clinical manifestations include deep vein thrombosis (DVT) and pulmonary embolism. Normal factor V interacts with activated protein C (APC) during the coagulation cascade. A point mutation (i.e., a single nucleotide change) in the factor V gene at codon 506 causes APC resistance. Codon 506 usually codes for arginine in Factor V protein, the site that normally interacts with APC. APC acts normally to inactivate the pro-coagulating activity of Factor V by cleaving it at that site.

In the mutant Factor V protein, a glutamine is present at that site and blocks the interaction with APC; thus inactivation of Factor V’s coagulating activity goes unchecked. The patient is said to be APC-resistant.

Humans have 2 copies of this gene (each copy is called an allele). Patients with one or both alleles of Factor V gene mutated, have a resultant increased risk of thrombotic risk. The increased thrombotic risk for those heterozygous (1 allele affected) for the mutation, also called factor VLeiden is 5-10 times that of the normal population. The risk for homozygtes (2 mutant alleles) is 50-100-fold as great. Factor VLeiden is present at 2-7% in the general population and is the most common cause of familial thrombotic syndromes.

PCR amplification of the region in the factor V gene containing the mutation is the basis for the common molecular diagnostic assay. Amplification of the target sequence is followed by restriction enzyme analysis to detect the single base pair mutation. The restriction enzyme digestion of the amplified PCR product generates distinct fragments for normal, heterozygous, and homozygous individuals for this gene. These fragments are electrophoresed to visualized and identify the status of this genetic locus in each individual.

Highlights of the methodology:
  1. Genomic DNA is extracted from patient’s whole blood.
  2. DNA is put into a PCR reaction containing primers for a 223 base pair fragment of the Factor V gene that encodes the region of the gene that corresponds to codon 506 in the final protein product.
  3. An amplified product (i.e., amplicon) of 223 base pairs results.
  4. This amplified product is then digested with the restriction endonuclease Mnl I.
  5. After digestion, the restriction digests are electrophoresed in an agarose gell to separate restriction fragments by size.
  6. Analysis of restriction fragments is performed and presence of Factor VLeiden can be determined for each individual.

PCR amplification produces a 223 base pair fragment (linear DNA sequence).

In normal individuals, the Mnl I enzyme cuts this 223 bp fragment twice to produce 3 different fragments:

a 82 base pair fragment; a 37 base pair fragment; and a 104 base pair fragment

 

 

82 37 104

 

 

 

Mnl I recognizes gAgg, thus when a mutation (gCgAggAA to gCAAggAA) occurs in that sequence a restriction site is lost.

Below is the gel electrophoretic results from 6 patients (Lanes 1 – 6). In lane M, there is a marker containing 50 bp and 150 bp fragments. In lane Neg, the negative control reaction sample was loaded. Please note that the 37 bp band can not be seen in the gel below. In lanes 2 – 6 the lowest band is the 82 bp band. Please interpret the results in the gel shown.

In which lane is the normal patient’s sample?

In which lane is the heterozygous patient’s sample?

In which lane is the homozygous MUTANT patient’s sample?

How many base pairs long is the top band in lanes 1 and 2?