Curtain open

In 1983, James Gusella discovered the genetic basis for Huntington's Disease. Called the Huntingtin (HTT), it was "the first disease-associated gene to be molecularly mapped to a human chromosome."^[1]

Readers of my posts might wonder at that relatively late date. 1983? Genes were being discovered and mapped in peas and flies and worms long before that. What took humans so long?

Well, you see, it takes two to tango. I've talked extensively about the molecular side of things in prior posts — the molecular tango partner. But you also need a phenotype — the macroscopically visible product of the gene — and humans are not as easy as yeast and Drosophila to partner up and force to have experimental children for the sake of clarifying phenotypes. To put it more scientifically, "informative crosses [were] lacking"^[2]. Population samples were also too small to be statistically significant. And lastly, the human genome is enormous compared to these model lab organisms.

This is not what I mean by "model lab".

_{Source: Maxpixel FreeGreatPicture}

In other words, the science was up to the task — molecularly speaking. But whereas it was relatively easy to cross flies and get phenotypes such as curly wings and then ask what genes were responsible for them, it wasn't as easy to isolate a trait of interest in humans. And without a clear trait of interest, you can't go on a gene hunt for that trait.

So how did they do it?

RFL(P?)

It's uncertain whether rolling on the floor laughing was in existence in the 80s, but restriction fragment length polymorphisms (RFLPs) certainly were. These are neutral DNA polymorphisms (differences in DNA letters). Neutral because their being different doesn't matter. They are silent variations, if you will, between individuals, that are not associated with any macroscopically observable differences in the individual.

They're important because this is how the problem of "no clear phenotypes" was solved. Like Sturtevant did in his famous all-nighter, you could build a linkage map (also called a chromosome map) in flies, because you had clear visible traits like curly wings and red eyes — and in some cases unmissably visible like feet in place of a fly's antennae (called, aptly, antennapedia).

Biologist David Botstein's idea was, instead of using phenotypes to build linkage maps, to use these polymorphisms instead.^[3] All you needed really was some kind of marker or road sign, and these polymorphisms served perfectly.

Mind you, another method of gene-finding, namely doing complementation tests, was out of the question for Huntington's disease, because complementation relies on dominant genes "rescuing" recessive genes, and Huntington's was dominant, so nothing could rescue it.

I didn't put that name there. The man wears his name on his sleeve.

_{Source: Wikimedia Commons contributors}

So the first hurdle being thusly overcome by Botstein, it was up to other long-legged humans (partly Gusella himself) to jump over the hurdle of small population size. This was solved by finding two Venezuelan villages in which the prevalence of the disease was unusually high: more than 18,000 people, to be inexact, most descended from a single family.^[4] Heading the study was Gusella's paper's second named author Nancy Wexler, who continued the research for 20 years, until political events forced her to discontinue this remarkably longitudinal study.^[7]

Last hurdle

Fork needs to be taught some manners.

_{Source: purdue.edu}

So now James Gusella inherits a 3-pronged problem with 2 prongs filed off. One prong remains: the human genome is vast. It's marked alright, like a ruler, due to Botstein's work. But it's still vast: how is Gusella going to find the marker that is nearest the gene responsible for Huntington's?

Remember, there's no human genome map yet, the Human Genome Project hasn't yet begun.

So Gusella had this bright idea: he'll take pieces of DNA, see if they contained a Botstein polymorphism, and then check to see if it was linked to Huntington's. Yes, that's the opposite of a bright idea. It's the absence of an idea. Gusella basically decided to brute-force the thing.

Given that there's around 3 million Botstein markers in the human DNA, Gusella was looking at a long night.

So of course he found the site he was looking for at his 12th try.

Wonder if he ever tried playing the Lotto.

Hunting the Huntingtin gene: Gusella's Lucky Number 12

Lucky number. Unless of course you can't see it, in which case that's unlucky, boy this joke went south real fast, I apologize.

_{Source: Wikimedia Commons contributors}

On his 12th trial, Gusella got the link. Lucky right?

But that wasn't enough. He's still not on the spot itself, just near it. A 1% recombination would mean he's a million bases (letters) away from the gene itself. I'm not pulling that number our of a magic hat. Later, when the same method was being used to discover the gene for cystic fibrosis, they got within 1.5 million letters:

The CF gene was thought to be inside this region [...] But between these markers lay 1.5 centimorgans (map units) of DNA, a vast uncharted terrain of 1.5 million bases.^[2]

The Huntington gene turned out to be 4Mb (4 million bases) away from the Lucky Number 12 locus.^[7]

So what could Gusella do in this case?

He could keep trying the random method, that's one option. But the probabilities were against him. He must proceed from where he was, somehow do it in a directed manner this time.

To the rescue comes a technique called physical mapping. What you do basically is you take the DNA pieces you've made via cloning, you radioactively label them, then you swish them around (I'm oversimplifying) in a container containing your fragment that is linked to the Huntington's gene, and hopefully the fragments that overlap will stick to each other. And now you can do the same with the longer fragment, and hopefully get closer to the gene. I say hopefully, because you could also get farther away from it. But eventually, by testing linkage, you'll know which of the two directions is the correct one. And you will gradually "walk" toward your gene of interest, in what's called, unsurprisingly, chromosome walking.

Is that the end? No. Even at 0% recombination, it doesn't mean that whole region of DNA is the gene responsible for the disease in question. Your children may inherit your diseases along with your property, but that doesn't mean the two are connected.

So what you do is you keep testing people, populations, and gradually arrive at what is most probably the sequence of letters that causes the disease you are looking for.

_{Source: flickr, modified}

In the case of Huntington's, a series of repeated letters CAG were found to be the culprit. These are the Huntingtin gene. Between 7 and 35 of these repeats are normal and will leave you unaffected, 36 to 40 CAG repeats may or may not give you the disease, and more than 40 repeats means you are royally CAG-rewed.^[6]

Curtain close

Most call him Vitruvian Man. I call him Jumping Jack.

_{Source: Wikimedia Commons contributors}

Imagine every researcher having to do what Gusella did with each individual gene of interest. That's what the Human Genome Project was meant to render unnecessary. A common DNA library open to all scientists for free, a complete map of the human genome, so that the scientists didn't each have to spend years upon years doing the same painstaking work.

After 10 years of chromosome walking (not so lucky now, eh?), in 1983 Gusella published his work on the HD gene, and in 1984 the idea of the Human Genome Project was picked up by the US government. The project was launched in 1990 and in 2000 the whole human genome was sequenced. 10 years for a single gene. 10 years for the whole genome. That's what collaboration will do for you.

REFERENCES

^{1. Chial, H. (2008) Huntington's disease: The discovery of the Huntingtin gene. Nature Education 1(1):71 https://www.nature.com/scitable/topicpage/huntington-s-disease-the-discovery-of-the-851}

^{2. Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000. Structural genomics. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21841/}

^{3. Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics. 1980;32(3):314-331. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1686077/}

^{4. Gusella, J. F., et al. A polymorphic DNA marker genetically linked to Huntington's disease. Nature volume 306, pages 234–238 (17 November 1983) doi:10.1038/306234a0 https://www.nature.com/articles/306234a0}

^{5. Wikipedia contributors, "Primer walking," Wikipedia, The Free Encyclopedia, https://en.wikipedia.org/w/index.php?title=Primer_walking&oldid=823994568 (accessed February 16, 2018).}

^{6. Wikipedia contributors, "Huntingtin," Wikipedia, The Free Encyclopedia, https://en.wikipedia.org/w/index.php?title=Huntingtin&oldid=809414625 (accessed February 16, 2018).}

^{7. Bates, G. P. History of genetic disease: The molecular genetics of Huntington disease — a history. Nature Reviews Genetics 6, 766-773 (2005) doi:10.1038/nrg1686. https://www.nature.com/scitable/content/History-of-genetic-disease-The-molecular-genetics-15297}

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Earlier Introduction to Biology episodes:

14: Sequencing DNA using Gel Electrophoresis

13: Cutting, Pasting, Cloning, and GMOs

12: How a Boy From an Indian Village Broke the Code of Life and Won the Nobel Prize

11: The Most Beautiful Experiment In Biology

10: The Great GATC-by: The Most Famous Science Paper of the 20th Century

9: The Great Kitchen Blender Experiments: How DNA was proved to be the seat of heredity

8: Finding, Counting, and Ordering Genes Using Incredibly Sophisticated Biomolecular Megatechnology

7: Christmas Disease — Yes, it's real, 100% scientifically proven!

6: The Most Famous All-Nighter in the History of Genetics

5: Mendel's Lucky Number Seven — The law of genetics that almost wasn't

4: How Cells Use Logic To Do The Impossible

3 : Armchair Science — The Discovery of Proteins' Secondary Structure

2 : How Cell Membranes Form Spontaneously

1 : Eduard Buchner: The Man Who Killed Vitalism

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