Rare Disease Day 2013: cystic fibrosis

Last week I shared two of the many slide decks I created for the sophomore Integrated Sciences biology course (ISC235/236), which I taught at Princeton from 2010 to 2012. Each slide deck required one to two days of Google and PubMed sleuthing followed by artful assembly in Powerpoint. In honor of Rare Disease Day 2013, which is today, I’m inaugurating the process of sharing and annotating all my Princeton-era teaching slide decks with this post on cystic fibrosis. Feel free to reuse, revise, or re-imagine.

The people who inherit two broken copies of a single gene called CFTR get cystic fibrosis. The CFTR gene was discovered (“cloned”) in 1989 at a cost of almost $200M. But it took 23 years for the first genuine breakthrough drug called Kalydeco/ivacaftor to be developed for only a subset of people with cystic fibrosis (CF) harboring a particular mutant form of the CFTR gene called the Celtic allele. For some perspective, the discovery of CFTR predates my former students by several years, many of whom weren’t even aware of the disease.

I opened the slide deck with a karyotype. Most students quickly realized that it yielded several clues about the person who supplied these chromosomes, namely this person is a male with Down Syndrome:

But cystic fibrosis isn’t caused by having an extra chromosome, which is easy to spot in karyotype. The mutations that cause CF are tucked away in a single gene called CFTR, which encodes a chloride ion transporter protein that is expressed in the epithelial tissue of the lungs. Some day we may be able to visualize point mutations microscopically with some future version of the iPhone, but we can’t today and geneticists weren’t able to 25 years ago.

The recombinant DNA revolution that swept across genetics in the 1970s presented a solution to the problem of finding a needle (a mutation) in a haystack (a chromosome) of haystacks (a genome, or 46 chromosomes). David Botstein and colleagues published a wonderful paper that’s been cited almost 6,000 times on a set of molecular techniques that now seem quaint in comparison to next-gen sequencers. Restriction fragment length polymorphisms (RFLPs) are like fingerprint patterns that, in the limit, distinguish any two people in the world:

With RFLPs, geneticists could link specific banding patterns with a disease in sick people. But back in the day, it wasn’t easy to shuffle around chromosomes, which had to be propagated and amplified by living cells and then chemically extracted and manipulated in the lab. Somatic cell hybrids presented a crude albeit effective way to isolate chromosomes of human DNA against a backdrop of non-human genome, e.g., mouse, in search of the one chromosome that contained polymorphisms linked to cystic fibrosis.

And so the hunt began. By 1985, the year Michael J. Fox was lighting up the silver screen in Back to the Future, the CF-causing gene was tracked down to a chromosome. But there isn’t just one singular CF-causing mutation. In fact, an individual with CF may not even have inherited the same exact broken version of CFTR from both parents. Turns out the majority of people with CF have a mutant allele of CFTR in which 3 DNA bases are deleted, ∆F508. Here’s the original table from the paper proving that a polymorphism called DOCRI-917, which is present in people with CF, resides on chromosome 7:

How exactly do CFTR mutations break the CFTR protein? CFTR like many channels and transporters, resides in the plasma membrane, or at the cell surface. But it isn’t created there, rather it is synthesized inside the cell and must then wend its way to the cell surface via vesicular transport pathways, which shuttle cellular components from one location to another inside tiny compartments called vesicles. Mutant CFTR can’t make its way to the surface, where they normally accumulate:

This gave researchers the idea that if they could find “pharmacological chaperones” that would suppress the misfolding effects of the mutation on the CFTR protein structure, then CFTR would survive the quality control system and make its way to the cell surface. This idea bore fruit with Kalydeco:

Right now clinical trials are underway with Kalydeco and another compound that may restore the function of ∆F508, which in the US is half of the people with CF.