Abstract
Designing a diagnostic test to determine the presence of the N1303K mutation
of the CFTR gene and is imperative to the proper identification and treatment of Cystic Fibrosis. The PCR assay designed in this research can identify a DNA template as wildtype, just as similar PCR assays
have been implemented for other mutations (Friedman et. al., 1991). PCR primers in this research were designed to distinguish
between N1303K mutated and wild-type DNA sequences. DNA templates were wildtype
human bronchial epithelial cells and plasmids containing the mutated CFTR cDNA sequence. DNA
was purified and extracted, PCR was performed and the results of the individual PCR assays were interpreted using agarose
gel Electrophoresis. Expected band lengths, based on the known CFTR sequence,
were 516 bp for the wildtype sequence and 622 bp for the mutant. Results
of the testing were inconclusive. Only the wildtype primer produced banding when used with the wildtype template. However
both the mutant-seeking and wild-type seeking primers bound to the N1303K mutated cDNA.
Concurrently, research was done into possible selective pressures leading to
the modern prevalence of CFTR mutations. The hypothesis was that multiple selective pressures could lead to the observed frequency
of CFTR mutations. Vibrio cholerae, Mycobacterium
tuberculosis and Salmonella enterica, were all believed to be possible selective
pressures (Poolman and Galvani, 2006). Through review of research articles as well as historical evidence, it was determined
that all three diseases were viable selective pressures, but of different strengths based on a fundamental set of criteria.
Discussion
N1303K
is a genetic mutation that inhibits multiple organs from functioning properly by altering the folding of the CFTR protein
which acts as an ion channel in epithelial cells. Presence of N1303K can be determined in a sample of DNA by performing PCR
to amplify a targeted sequence of the DNA and interpreting the results of the PCR on an agarose gel electrophoresis (Wittwer
et al., 1993). If a patient has an N1303K mutated allele, then there will be
a polymorphism on the 4041st nucleotide base of their CFTR gene. This
polymorphism can be detected using PCR by specially designing a set of forward and reverse primers (Liu et al, 2001).
Testing
of designed primers was performed through PCR on wildtype genomic DNA. Because of several successive events of nonspecific
binding, the annealing temperature was increased in intervals of 2°C from 54°C to a temperature of 58°C. A higher annealing
temperature corresponds to a decrease in non-specific binding because at a higher temperature more bonds are required for
the primer to remain annealed to the template sequence, thereby reducing the chance of the primer binding to the wrong areas
or creating hairpins (Wittwer et al., 1993). Conclusive results were achieved at 58°C, with
a 516 bp band in the wildtype reaction lane confirming that the template was wildtype.
Testing
on the mutant N1303K plasmid DNA was inconclusive. Both the wildtype and mutant primers annealed to the plasmid template,
yielding identical banding in both tests. These results are due to the fact that primers are annealing to the target area
whether or not the mutation is present, as single nucleotide polymorphisms are easier for Taq to bridge than larger mismatches
such as F508. However our evidence does not support this theory, as the existence of proper results with wildtype DNA suggests
(Figure 2). Raising the annealing temperature or stringency could resolve the
situation, since a primer is less likely to bind unless all of its bases match the template.
Another
potential explanation of results is the realization that wildtype testing was done on genomic DNA while mutant testing was
done on cDNA. This required two separate reverse primers but did not affect the
forward primers because their complement sequence was located on an exon of the CFTR gene. Since the forward primers distinguished
between the mutant and wildtype alleles this was not anticipated to be a problem, regardless the results were contradictory
to expectations. Future investigation dealing with this subject would focus on
yielding specific results with either only mutant-seeking or wild type-seeking primers.
The PCR assay developed in this research cannot
yet be confirmed as a viable diagnostic method because conclusive positive results with mutant DNA had not been achieved.
The fundamental approach is supported by the fact that diagnosis of similar genetic mutations has been performed by other
researchers using the same method (Friedman et al., 1991). Once the test is deemed
viable, it could be used to screen individuals for the N1303K-CFTR mutation.
Examination of Selective
Pressures for Cystic Fibrosis
The three criteria used to determine if the diseases being studied were plausible
selective forces were; molecular, clinical, and historical lines of evidence. Tuberculosis
and cholera fulfilled two of the three criteria, while typhoid fever only fulfilled one.
The difference in each disease’s ability to fulfill the three criteria has been studied thoroughly.
On the molecular level the pathogenesis
of the three different bacteria results in three different theories of heterozygote advantage.
Vibrio cholerae and Salmonella enterica
are both enteric diseases which act directly on CFTR channels in the small intestine (Barua and Greenough, 1992; Pier et al.,
1998). Virbrio cholera creates a salty
conditions in the small intestines of its host by releasing an enterotoxin which through a series of biochemical reactions
results in the elevation of cyclic adenosine monophosphate (cAMP) which locks ion channels dependent on ATP kinase for function
(including CFTR) into the open position (Barua and Greenough, 1992). The open
ion channels cause a mass exodus of chloride ions into the lumen of the small intestines, which in turn results in the secretory
diarrhea that is characteristic of Cholera (Barua and Greenough, 1992). If a
person is heterozygous for Cystic Fibrosis they will have less functional CFTR channels, effectively decreasing the potential
for secretory diarrhea. Salmonella enterica
uses the CFTR ion channels in the epithelium of the small intestines for direct entry into enter a host. If there are a reduced number of CFTR channels then the bacterium will be less able to invade its host
and become virulent (Pier et al., 1998).
Tuberculosis differs from Cholera and Typhoid fever as it is a disease of the
respiratory tract. Virulence of Mycobaterium
tuberculosis is shown to correlate with the bacterium’s ability to thrive inside the phagosome of host macrophages
(Tobacman, 2003). It is theorized that Mycobaterium
tuberculosis’ ability to thrive within the phagosome relies on the presence of the lysosomal enzyme Arylsulfatase
B (Tobacman, 2003). It has been shown that carriers of the Cystic Fibrosis allele
have markedly increased mucopolysaccharide production, possibly a result of decreased Arylsulfatase B activity, which would
normally digest these polysaccharides (Danes et al 1974). Recent experiments
have shown an increase in Arylsulfatase B activity when CFTR is corrected in cells in
vitro (Bhattacharyya et al., 2007). However
evidence of experimentation that links increased mucopolysaccharide levels and a diminished ability for Mycobacterium tuberculosis to survive in a host was never discovered. A
contradictory line of evidence points to high salt levels in the lung decreasing the ability for a host bactericidal factor
to fight off invading bacterial infections (Smith et al., 1996).
Clinical studies of patients affected with Cystic
Fibrosis have shown a decreased incidence of infection by the bacterium Mycobacterium
tuberculosis (Smith et al, 1984; Kilby et al., 1992). No clinical evidence
has been uncovered that links the presence of the CF allele to a decreased infection rate of Salmonella enterica. Clinical evidence of Cholera is inconclusive. One
study found that there was a difference in chloride secretion in mice heterozygous for CF and affected with Vibrio Cholera (Gabriel et al., 1994), while another study found that there was no measurable difference (Cuthbert
et al., 1995).
Historical evidence revealed a distinct difference
in each disease’s individual affect on the greater European population. Tuberculosis
was clearly the most devastating disease, appearing the earliest (1600’s), and maintaining the highest death rate over
the longest period of time (Poolman and Galvani, 2006). Cholera was second in
its affect on European populations. Cholera epidemics affected Europe in waves,
first hitting the continent in 1817 and continuing through to the early 1900’s (Barua and Grennough, 1992). Typhoid outbreaks were more intermittent then Cholera and more localized usually affecting single cities
at a time (Hays, 2005). Tuberculosis death rates commonly grew to 750 deaths/
100,000 people in cities throughout Europe. Cholera at its worst had death rates
of 300deaths/ 100,000 people, and Typhoid fever had common death rates of 50-100 deaths/100,000 people (Hays, 2005).
Evidence from the research thus proves that no single
disease meets all criteria for the necessary lines of evidence to explain their ability as a viable selective pressure. Tuberculosis has been nominated as the strongest selective force because of its ability
to maintain high death rates over a sustained period of time. Both Cholera and
Typhoid fever were too intermittent and did not kill enough of the European population to be the sole selective pressures. However this doesn’t mean that Cholera and Typhoid could not select for the
Cystic Fibrosis allele, only that tuberculosis was the strongest selective force because of its widespread dissemination throughout
European populations. A good example of this is the observed frequency of CFTR
mutations in North Africa. Tuberculosis did not reach Africa until the late nineteenth
century, yet there is a high frequency of the N1303K and G542X mutations in this region (Poolman and Galvani, 2006; Estivill
et al., 1997). Both Cholera and Typhoid fever were endemic in North Africa prior
to the nineteenth century, and thus we hypothesize they acted as the primary selective forces for the Cystic Fibrosis allele
in that area (Barua and Greenough, 1992; Crump et al., 2004).
Future Directions
Future studies would further investigate if the
role of an Arylsulfatase B deficiency in CF patients is linked to Mycobacterium tuberculosis’s
ability to thrive within its host. Also further investigation could follow the
relative frequencies of certain CFTR mutations in particular ethnic groups, such as the W1282X mutation in Ashkenazi Jewish
populations, in order to determine if allele frequency is attributed to heterozygote advantage or a founder effect/ cultural
norms of the population.
The inconclusive results of PCR on mutant cDNA samples must be further investigated.
The test may be replicating fragments of the plasmid other than the target sequence, or the polymerase is disregarding
the single nucleotide polymorphism and synthesizing the target region of the template DNA regardless of the presence of the
mutation. Additional tests including sequencing and RFLP could determine whether the proper sequence is being replicated.
