Every single person on Earth can claim to have been born at some point. In reality,
if you were born in a hospital in Victoria, Australia in 1989 or later,
you would probably be able to claim you underwent a heel-prick test as well. A heel-prick test is a
clinical examination performed on newborn babies that screens for rare diseases,
allowing newborns a better chance of receiving an early diagnosis, as well as improving the ease of
genetic counselling for affected families who are at high risk of having a genetic disorder.
You may have wondered what was the significance of mentioning 1989 Victoria. Well, it signifies the
addition of cystic fibrosis as a new condition to the heel-prick test screen. Cystic fibrosis
(or CF) is an autosomal recessive genetic disorder brought on by germline mutations.
It affects the cells responsible for production of mucus, sweat, and gastrointestinal secretions.
Like all recessive conditions, CF requires two copies of the defective CF gene,
one copy inherited from each parent and with both parents carrying at least one copy of
the defective gene. The fusion of genes results in the production of four gametes with infected
individuals expressing both recessive alleles. Statistically this represents
a 25 percent chance that the child will or will not have CF and a 50 percent chance they
will be a carrier but will not be affected. According to the journal of cystic fibrosis,
a conservative estimate places the diseases current global prevalence at 162,000 people.
Unsurprisingly, with such clinical importance,
there was naturally a lot of effort put forwards to pinpointing the gene responsible for CF and
defining its protein product. In a series of three studies published in 1989 – the
same year that CF screening was introduced in Victoria – the CF gene was discovered.
In order to understand the basic defect in CF, cloning of the gene and definition of its protein
product was required. One of the three studies, led under John R. Riordan and his team, postulated
that they might locate the CF gene by isolating and characterizing cDNA clones. This eventually
led to the identification of the cystic fibrosis transmembrane conductance regulator, or CFTR.
A cross-species gene expression study served as the first step in the isolation of cDNA
clones. Using a prior study that localised the CF locus to the extended arm of chromosome 7,
significantly simplified the comparison between
DNA sequences that shared the same identity with the relevant CF allele.
Through the use of two CF-positive DNA segments that looked for cross-species
hybridization signals, a non-CF cDNA library was investigated.
From this library, a pertinent clone was isolated to one of the DNA segments.
Unfortunately, the shared cDNA sequence was short, only 113 base pairs. Thus,
further cDNA library screening was required in order to obtain a more comprehensive set of the
coding region to better match the estimated size of the CF transcript at 6.5 kilobases.
In accordance, several more screenings were performed making the new clone total 18. With
this a contiguous coding region of the CF locus could be created through overlapping all cDNA
clones together. The consensus created contained an open-reading frame that could encode a protein
1,480 amino acids large within a 6.1 kilobase long sequence. Strangely, this was significantly
less than predicted based on the projected 6.5 kilobase transcript from the RNA hybridization
tests. However, this discrepancy was attributed to the presence of aberrant structures which
were preferentially retained while proper clones were lost during interpretation.
cDNA was further characterised by subsequent gel hybridization experiments with genomic DNA.
The results indicated multiple restriction fragments across several cDNA clones,
signalling the presence of multiple exons within the putative CF gene. For the first time,
the exon-intron boundaries on the verified cDNA clones could be taken, and identification of 24
exons within a gene locus extending roughly 250 kilobases, was made. Unfortunately,
the consensus sequence used was later found incomplete. Missing DNA fragments caused by
the presence of exons therein were refractory to isolation by hybridization due to their small
size, or skipped over during cDNA cloning. Later studies amended the amount from 24 exons to 27,
as well as establishing the full-length of the CFTR mRNA to contain 6,128 nucleotides.
Through comparison of the completed CF sequence against unaffected individuals,
detection of the mutation was now also possible.
Their most striking results was the presence of a deletion mutation that
resulted in the loss of 3 -base pairs that coded for phenylalanine in the predicted CF polypeptide.
Generation of the predicated polypeptide was also used to describe the structural characteristics
of the proteins. Riordan et al. identified two of the proteins most distinctive motifs,
consisting of a domain with properties consistent with membrane association,
and a sequence domain believed to be involved in ATP binding. The justification of these traits
was that the predicted CF gene was likely involved in the transport of ions across the
membrane because of its striking similarities to numerous other membrane-associated proteins,
including P-glycoproteins and several cation channel proteins.
These advancements made in establishing the basis of cystic fibrosis genetics
paved the way for more complex genome-wide association studies and focused research on
elucidating the precise mechanism underlying the pathophysiology of cystic fibrosis. Furthermore,
work in identifying the CFTR gene and its genetic product further
validated the use of complementary cloning as a characterisation technique for unknown genes.