Category Archives: Medicine in general

Has the great white whale of oncology finally been harpooned?

The ras oncogene is the great white whale of oncology. Mutations in 20 – 40% of cancer turn its activity on so that nothing can turn it off, resulting in cellular proliferation. People have been trying to turn mutated ras off for years with no success.

A current paper [ Cell vol. 165 pp. 643 – 655 ’16 ] describes a new and different way to attack it. Once  ras is turned on (either naturally or by mutation) many other proteins must bind to it, to produce their effects — they are called RAS effectors, among which are the uneuphoniously named RAF, RalGDS and PI3K. They bind to activated ras by the cleverly named Ras Binding Domain (RBD) which has 78 amino acids.

The paper describes rigosertib, a not that complicated molecule to the chemist, which inhibits the binding (by resembling the site on ras that the RBD binds to). It is a styryl benzyl sulfone and you can see the structure here —

What’s good about it? Well it is in phase III trials for a fairly uncommon form of cancer (myelodysplastic syndrome). That means it isn’t horribly toxic or it wouldn’t have made it out of phase I.

Given the mechanism described, it is possible that Rigosertib will be useful in 20 – 40% of all cancer. Can you say blockbuster drug?

Do you have a speculative bent? Buy the company testing the drug and owning the patent — Oncova Therapeutics. It’s quite cheap — trading at $.40 (yes 40 cents !). It once traded as high as $30.00 — symbol ONTX. I don’t own any (yet), but for the price of a movie with a beer and some wings afterwards you could be the proud owner of 100 shares. If Rigosertib works, the stock will certainly increase more than a hundredfold.

Enough kidding around. This is serious business. In what follows you will find some hardcore molecular biology and cellular physiology showing just what we’re up against. Some of the following is quite old, and probably out of date (like yours truly), but it does give you the broad outlines of what is involved.

The pathway from Ras to the nucleus

The components of the pathway had been found in isolation (primarily because mutations in them were associated with malignancy). Ras was discovered as an oncogene in various sarcoma viruses. Mutations in ras found in tumors left it in a ‘turned on’ state, but just how ras (and everything else) fit into the chain of binding of a growth factor (such as platelet derived growth factor, epidermal growth factor, insulin, etc. etc.) to its receptor on the cell surface to alterations in gene expression wasn’t clear. It is certain to become more complicated, because anything as important as cellular proliferation is very likely to have a wide variety of control mechanisms superimposed on it. Although all sorts of protein kinases are involved in the pathway it is important to remember that ras is NOT a protein kinase.

l. The first step is binding of a growth factor to its receptor on the cell surface. The receptor is usually a tyrosine kinase. Binding of the factor to the receptor causes ‘activation’ of the receptor. Activation usually means increasing the enzymatic activity of the receptor in the tyrosine kinase reaction (most growth factor receptors are tyrosine kinases). The increase in activity is usually brought about by dimerization of the receptor (so it phosphorylates itself on tyrosine).

2. Most activated growth factor receptors phosphorylate themselves (as well as other proteins) on tyrosine. A variety of other proteins have domains known as SH2 (for src homology 2) which bind to phosphorylated tyrosine.

3. A protein called grb2 binds via its SH2 domain to a phosphorylated tyrosine on the receptor. Grb2 binds to the polyproline domain of another protein called sos1 via its SH3 domain. At this point, the unintiated must find the proceedings pretty hokey, but the pathway is so general (and fundamental) that proteins from yeast may be substituted into the human pathway and still have it work.

4. At last we get to ras. This protein is ‘active’ when it binds GTP, and inactive when it binds GDP. Ras is a GTPase (it can hydrolyze GTP to GDP). Most mutations which make ras an oncogene decrease the GTPase activity of RAS leaving it in a permanently ‘turned on’ state. It is important for the neurologist to know that the defective gene in type I neurofibromatosis activates the GTPase activity of ras, turning ras off. Deficiencies (in ras inactivation) lead to a variety of unusual tumors familiar to neurologists.

Once RAS has hydrolyzed GTP to GDP, the GDP remains bound to RAS inactivating it. This is the function of sos1. It catalyzes the exchange of GDP for GTP on ras, thus activating ras.

5. What does activated ras do? It activates Raf-1 silly. Raf-1 is another oncogene. How does activated ras activate Raf-1 ?  Ras appears to activate raf by causing raf to bind to the cell membrane (this doesn’t happen in vitro as there is no membrane). Once ras has done its job of localizing raf to the plasma membrane, it is no longer required. How membrane localization activates raf is less than crystal clear. [ Proc. Natl. Acad. Sci. vol. 93 pp. 6924 – 6928 ’96 ] There is increasing evidence that Ras may mediate its actions by stimulating multiple downstream targets of which Raf-1 is only one.

6. Raf-1 is a protein kinase. Protein kinases work by adding phosphate groups to serine, threonine or tyrosine. In general protein kinases fall into two classes those phosphorylating on serine or threonine and those phosphorylating on tyrosine. Biochemistry has a well documented series of examples of enzymes being activated (or inhibited) by phosphorylation. The best worked out is the pathway from the binding of epinephrine to its cell surface receptor to glycogen breakdown. There is a whole sequence of one enzyme phosphorylating another which then phosphorylates a third. Something similar goes on between Raf-1 and a collection of protein kinases called MAPKs (mitogen activated protein kinases). These were discovered as kinases activated when mitogens bound to their extracellular receptors.There may be a kinase lurking about which activates Raf (it isn’t Ras which has no kinase activity). Removal of phosphate from Raf (by phosphatases) inactivates it.

7. Raf-1 activates members of the MAPK family by phosphorylating them. There may be several kinases in a row phosphorylating each other. [ Science vol. 262 pp. 1065 – 1067 ’93 ] There are at least three kinase reactions at present at this point. It isn’t known if some can be sidestepped. Raf-1 activates mitogen activated protein kinase kinase (MAPK-K) by phosphorylation (it is called MEK in the ras pathway). MAPK-K activates mitogen activation protein kinase (MAPK) by phosphorylation. Thus Raf-1 is actually mitogen activated protein kinase kinase kinase (sort of like the character in Catch-22 named Junior Junior Junior). (1/06 — I think that Raf-1 is now called BRAF)

8. The final step in the pathway is activation of transcription factors (which turn genes off or on) by MAP kinases by (what else) phosphorylation. Thus the pathway from cell surface is complete.

Is that mutation significant?

Face it, our genomes are a real mess. A study of just the parts of the genome coding for amino acids (2% at most) in about 2,500 people found an average of 205 variants which change the amino acid coded for IN EACH PERSON. Each person also had an average of 3 termination codons in the 15,000+ protein coding sequences they studied. So they are wandering around with 3 abnormally short proteins. You can read more about it in this old post –

Here’s the problem — these people were healthy. Obviously, not a problem for them, but a big problem for physicians attempting to do genetic counseling. For how it affected epilepsy counseling see —

This brings us to Lynch syndrome (aka Hereditary NonPolyposis Colorectal Cancer — HNPCC). It is a familial cancer syndrome, and we now know what the problem is — mutations in any of four genes involved in a type of DNA mutation repair (there are many). The genes are called MSH2, MSH6, MLH1 and PMS2 (acronyms all whose names you don’t need to know) and the type of repair is called MisMatch Repair (MMR).

This isn’t academic at all. Suppose your aunt comes down with colon cancer and you get tested for mutations in one of the four, and a mutation is found. You’re fine now. The question before the house is — should you have your colon out? Colonoscopy won’t help because this kind of colon cancer doesn’t arise from polyps (which is what colonoscopy is looking for).

The problem is that the 4 genes are ‘peppered’ with missense variants (change the amino acid coded for). They are called VUS (Variants of Unknown Significance). The following paper [ Proc. Natl. Acad. Sci. vol. 113 pp. 3918 – 3820, 4128 – 4133 ’16 ] used a clever way to test a VUS for significance. This would have been impossible 5 years ago. What they did was use CRISPR to introduce the variant into the appropriate protein in mouse Embryonic Stem cells. Then they tested the manipulated stem cells for defects in MisMatch Repair. They tested 59 (yes fifty-nine) such VUSs and found that about 1/3 (19) produced MMR defects.

Fascinating time to be alive and reading about all this stuff.

Activating a proto-oncogene without mutating it

Many proto-oncogenes have to be mutated to cause cancer. Not so the TAL1, LMO2 genes. They drive blood formation, and are aberrantly activated (e.g. more proteins made from them is expressed) in T cell Acute Lymphoblastic Leukemia (TALL). [ Science vol. 351 pp. 1298- 1299, 1454 – 1458 ’16 ] activated them experimentally using the CRISPR technique, and therein hangs a tale.

Addendum 11 April — LMO2 is well known to gene therapists as early work (2002) using retroviruses inserted randomly in the genome to cure SCID (Severe Combined Immunodeficiency) resulted in TALL in 4kids.  The problem was that the vector integrated in multiple sites all over the genome and one such random site  turned on expression of LMO2.

I’ve written a series of six posts trying to imagine the incredible mass of DNA in a 10 micron nucleus on a human scale — we take it for granted, but it’s far from obvious how this is accomplished — here’s the link to the first — — just follow the links to the rest.

[ Cell vol. 153 pp. 1187 – 1189, 1281 – 1295 ’13 ] Hi-C and 5C (Carbon Copy Chromosome Conformation Capture) allow determination of chromatin organization and long range chromatin interactions in an unbiased genome wide manner at the megaBase scale. Topologically associated domains (TADs) are the way the genome in the nucleus is organized into megabase to submegaBase sized interacting domains. TADs are conserved between species and are invariant across cell types. [ Call vol. 156 p. 19 ’14 ] They average 700 – 800 kiloBases and are said to contain 5 – 10 protein coding genes and a few hundred enhancers. The expression of genes within a TAD is ‘somewhat correlated’. Some TADs have active genes, while others have repressed genes. Genomic interactions are strong within a domain, but are sharply depleted on crossing the boundary between two TADs.

Well TADs have to be separated from each other. The current thinking is that the boundaries are formed by sites in the DNA which bind the CTCF protein, and possibly cohesin proteins as well. CTCF is a large protein (although maddeningly I can’t seem to find out how many amino acids it has) with a molecular mass of 80 kiloDaltons. It’s DNA binding is quite specific as it contains 11 zinc fingers (each of which can specifically bind a 3 nucleotide stretch of DNA). In addition to binding to DNA it can bind to itself, forming a perfect way to form loops of DNA.

All the Science paper did was to delete a few CTCF binding sites using the CRISPR technique around the two oncogenes and bang — expression increased. Why?  Because the insulation between the TAD containing the genes and adjacent TADs was broken, allowing control of the genes by enhancers in the new and larger TAD that had been previously sequestered in an adjacent TAD.  The deletions were thousands of basepairs away from the coding sequence of the genes themselves.  All very nice, but it’s fairly artificial.

However the paper notes that across a large pan-cancer cohort, there was a 2 fold enrichment for boundary CTCF site mutations.

When knowledge isn’t power

Here is a genetic disease, where we’ve known exactly what’s wrong with the causative gene for 23 years, over 10,000 papers have been written (a Google search comes up with about 418,000 results (0.45 seconds), but we don’t know how the mutation causes the problems it does or have a clue how to treat the disease. So much for finding the cause of a genetic disease leading to therapy. Imagine how much harder cancer is.

I speak of Huntington’s chorea, and the causative gene huntingtin. It’s a terrible neurologic disease characterized by progressive movement disorders, dementia and incapacitation over a decade or two. Woodie Guthrie had it; fortunately Arlo escaped. Like many people with the disorder Woodie was quite fertile, having 8 children.

It being a neurologic disorder, I’ve read a lot about it, and my jottings about my readings over the past few decades have consumed 83,635 characters (aren’t computers wonderful)? I’ve had a fair amount of experience with it, as an Indian agent in Montana had it, and produced many progeny with his women, leading to a good deal of devastation in one tribe.

Neuron vol. 89 pp. 910 – 926 ’16 is an excellent recent review (but not one for the fainthearted). Several mysteries are immediately apparent.

First huntingtin is expressed in nearly every neuron, but only a few die. It is expressed outside the brain in lung ovary and testes, but they work just fine.

Second Huntingtin interacts with over 350 different proteins. Figuring which are the important ones has provided steady employment.

Third it exists in many forms, so many that there aren’t enough scientists living to test them all. This is because huntingtin is subject to a variety of chemical modifications (phosphorylation, ubiquitination, acetylation, palmitoylation, sumoylation) at FORTY-EIGHT different sites (listed in the article). So this gives 2^48 possible modified forms of the protein (either modification being present or absent). 2^48 = 281,474,976,710,656 if you’re interested.

In addition to the modifications, the protein is huge — some 3,144 amino acids occurring in 67 exons forming two mRNAs of 10,366 and 13.711 nucleotides.

Fourth The protein can also be chopped up by at least 5 different enzymes at 6 different sites, and some fragments are biologically active (toxic in tissue culture).

Naturally, the region with the mutation (near the amino terminal end) of the protein has been studied most intensively.

Huntingtin has its fingers in many physiologic pies — the reference is excellent in this area — these include vesicular trafficking, cell division, cilia formation, endocytosis, autophagy, gene transcription. Abnormalities of which one causes the neurologic disease.

The mutant form forms protein aggregates. Like Alzheimer’s disease senile plaque or the Lewy body of Parkinson’s disease, we don’t know if the aggregates are toxic or protective.

Fifth: Despite all its known functions we don’t know if the mutation produces a loss of some vital function of Huntingtin, or a new and toxic function.

Even worse, compared to cancer, Huntington’s chorea is ‘simple’ because we know the cause.

Is a rational treatment for Multiple Sclerosis in our future?

Two very recent papers taken together point the way to a rational treatment of multiple sclerosis (and probably all autoimmune disease). The short story:
Paper #1 found a way to find the antigen or antigens patients with MS are reacting to
Paper #2 found a way to selectively impair the response to an inciting antigen without clobbering the whole immune system

Some history: Some evening in 1966 or 1967 a fellow neurology resident and I were sitting on the ward having dealt with the complications of high doses corticosteroids for a case of optic neuritis (often the first sign of MS). I said, some day they’ll look at what we’re doing the way we look at docs of 200 years ago using leeches (and bloodletting). As a kid, I remember my parents driving into Philly. Shortly after getting over the Ben Franklin bridge we’d pass a pharmacy offering leeches on its sign.

It was obvious even back then that MS in some way was an attack by the immune system on the brain. Finding the particular antigen the system was reacting to would lead us to the cause and hopefully less simplistic treatment than clobbering the immune system. We didn’t know all the proteins we had or even how many, so people would look for antibodies to a variety causes (which they’d arrived at by reasoning, not data). Increased antibody titers to a variety of viruses were found, but that led nowhere. No one ever isolated a virus from MS brain, although sightings on electron microscopy were eagerly reported. Eventually it became obvious that the immune system was on high alert with increased antibodies to lots of things.

This leads to paper #1 [ Proc. Natl. Acad. Sci. vol. 113 pp. 2188 – 2193 ’16 ] To make a long story short they used something called the Human Protein Atlas Program to find what proteins the antibodies in MS patients were reacting to. So rather than having a theory about what MS patients might be reacting to and testing it, they looked at all proteins and watched. It’s the difference between being a Greek philosopher reasoning things out from first principles and collecting data. Only when the technology is available can you stop a priori theorizing and just look. Don’t be too hard on the earlier researchers, they didn’t have the tools.

The found that MS patients were reacting to a protein called anoctamin2, which actually showed increased expression near and inside the demyelinating plaques of MS.

For the gory details keep reading, otherwise skip to **** where I’ll discuss paper #2

Gory details — The Human Protein Atlas produces human protein fragments, selected on the basis of their low similarity to other proteins in the proteome. [ Science vol. 347 1260419 (23 Jan) ’15 ] The atlas hopes to find out where and how much of each our proteins is at the tissue and cellular level. It is based on antibody based profiling on tissue microarrays (of proteins?). This based on transcript expression (RNA-Seq), and immunohistochemistry (24,028 antibodies coresponding to 16,975 protein coding genes). 44 tissues were studied. The antibodies produced more than 13 million tissue based mmunohistochemistry images. They also report subproteomes (secreted proteins n = 3,171, and membrane bound proteins n = 5,570). Interstingly there was an overall concurrence between mRNA and protein levels for a given gene product across various tissues.

The PNAS paper profiled 2,169 plasma samples from MS cases and population based controls (with neurologic disease) using bead arrays built with 384 human protein fragments seleted from an initial screening with 11,520 antigens. There was increased reactivity to anoctamin2 (aka TMEM16B) in MS vs. controls (by how much?). This was corroborated in independent assay with alternative protein constructs and by epitope mapping with peptides covering the identified region of anoctamin 2.

ImmunoFLuorescence in human MS brain tissue showed increased anoctamin2 expression as small cellular aggregates near and inside MS lesions. The controls had other neurologic disease. There was a 5.3 fold change in fluorescence intensity in the MS group. The antibodies are directed against the amino terminal region.



Paper #2 — [ Nature vol. 530 pp. 422 – 423, 434 – 440 ’16 ] basically found a way to knock out the immune system’s response to a single antigen — not all of them. The point is that just an antigen by itself isn’t enough to turn the immune system on. A costimulatory molecule must also be present on the antigen presenting cell. If it isn’t there the immune system is actually turned off by forming regulatory T cells (which even though they are part of the immune system they actually turn it off).

One can form models of human autoimmune disease in mice. Two such are EAE (Experimental Allergic Encephalomyelitis) formed by giving the animal myelin basic protein (a constituent of myelin which is attacked in MS), and rheumatoid arthritis (formed by giving collagen to the animals). What is so great about this paper is that MHC II carrying peptides from collagen suppress disease in a mouse model of rheumatoid arthritis, but NOT in mice with EAE. MHC-II carrying CNS antigen peptides control EAE but not collagen induced arthritis.. In addition neither treatment impaired the immune response to infection — something that almost always happens when you clobber the immune system.

Well it’s a long way from the lab to the bedside, but imagine finding what the immune system is reacting to and stopping it (without stopping the immune system). That’s what these two papers portend. Exciting times.

Nicastrin the gatekeeper of gamma secretase

Once a year some hapless trucker from out of town gets stuck trying to drive under a nearby railroad bridge with a low clearance. This is exactly the function of nicastrin in the gamma secretase complex which produces the main component of the senile plaque, the aBeta peptide.

Gamma secretase is a 4 protein complex which functions as an enzyme which can cut the transmembrane segment of proteins embedded in the cell membrane. This was not understood for years, as cutting a protein here means hydrolyzing the amide bond of the protein, (e.g. adding water) and there is precious little water in the cell membrane which is nearly all lipid.

Big pharma has been attacking gamma secretase for years, as inhibiting it should stop production of the Abeta peptide and (hopefully) help Alzheimer’s disease. However the paper to be discussed [ Proc. Natl. Acad. Sci. vol. 113 p.n E509 – E518 ’16 ] notes that gamma secretase processes ‘scores’ of cell membrane proteins, so blanket inhibition might be dangerous.

The idea that Nicastrin is the gatekeeper for gamma secretase is at least a decade old [ Cell vol. 122 pp. 318 – 320 ’05 ], but back then people were looking for specific binding of nicastrin to gamma secretase targets.

The new paper provides a much simpler explanation. It won’t let any transmembrane segment of a protein near the active site of gamma secretase unless the extracellular part is lopped off. The answer is simple mechanics. Nicastrin is large (709 amino acids) but with just one transmembrane domain. Most of it is extracellular forming a blob extending out 25 Angstroms from the membrane, directly over the substrate binding pocket of gamma secretase. Only substrates with small portions outside the membrane (ectodomains) can pass through it. It’s the railroad bridge mentioned above. Take a look at the picture —

This is why a preliminary cleavage of the Amyloid Precursor Peptide (APP) is required for gamma secretase to work.

So all you had to do was write down the wavefunction for Nicastrin (all 709 amino acids) and solve it (assuming you even write it down) and you’d have the same answer — NOT. Only the totally macroscopic world explanation (railroad bridge) is of any use. What keeps proteins from moving through each other? Van der Waals forces. What help explain them. The Pauli exclusion principle, as pure quantum mechanics as it gets.

Bad news on the AIDs front

Bad news for those hoping for an AIDs cure. As you know, the active virus (HIV1) has a genome made of RNA. However, thanks to an enzyme it possesses called reverse transcriptase (which has led to Nobel prizes), it copies itself into DNA and integrates into the genome of lymphocytes. There it sits presumably doing nothing, but it’s always capable of activating and producing more infectious virus.

We seem to have fought the virus to a draw, using a cocktail of drugs which attack different aspects — HAART (Highly Active Antiretroviral Therapy). Success is usually considered being unable to detect viral RNA in the blood (see later). However blood cells are short-lived. What about the longer living lymphocytes found in the lymph nodes and spleen.

That’s what was studied in a current paper [ Nature vol. 530 pp. 5` – 45 ’16 ] but in only 3 people. All had no detectable virus in the blood (under 48 copies/milliLiter — an incredibly tiny amount — see later). What they did was to biopsy lymph nodes in the groin on study entry and at 3 and 6 months.

Then they sequenced the genomes of the lymphocytes from the nodes, to study the HIV1 DNA integrated into the genome. They found that the genome changed with time. This is very bad. Why?

Because it implies that, even though you the virus in the blood, the virus was not staying latent in the lymph nodes, but coming out of the lymphocytes and forming infectious virus which then mutated. Subsequently the mutated virus integrated into the genome of another lymphocyte. So even with what we consider excellent control, the virus is not purely latent. Drug resistance could arise from mutations (although they didn’t see it in this study).

Clearly, more people need to be studied this way (but serial biopsies? It will probably be done in prisoners, if such things are still done).

It’s worthwhile thinking about how incredibly selective and accurate our methods of analysis are. 48 copies of the viral RNA per milliLiter of blood is the lower limit of detection. Remember that water has a molecular weight of 18, so a liter of distilled water is 1000 grams / 18 grams = 55.5 Molar. A mole has 6 x 10^23 molecules. A milliLiter is 10^-3 liters. So 1 milliLiter of distilled water has 55 * 6 * 10^23 * 10^-3 == 3 * 10^22 molecules of water in it so the assay is finding 48 or more molecules of HIV1 RNA in the water haystack. Even figuring that the concentration of water in blood is 1/10 that of distilled water, this is still impressive.

The checklistization of medicine

Today at the ophthalmologists the assistant who prepped me by putting in eyedrops and checking my visual acuity, had to put some information in her computer. One of the questions was how long I’d been taking eye drops. I told her to look at the chart, since the information was already there. She asked me to guess, so I did, and she duly entered the guess, which is (probably) now in the (electronic) chart and certainly less accurate than what is already there. Such is the checklistization of medicine today. Thank God I’m retired. The ophthalmologist said it’s part of the software that insurance companies require.

My brother, who is still practicing internal medicine, now gets 20 (paper) sheets of mostly useless information for every ER visit of one of his patients. This includes
l. a sheet saying the patient did not fall off the gurney
2. attestation that the patient was treated in a culturally appropriate manner
3. attestation that the patient was given the opportunity to ask questions.
I’m not making this up, and neither is he.

He says the residents are complaining that less time is available for the patient due to all this.

I guarantee you that this malarkey was not put in at the request of physicians and nurses actually attempting to take care of people.

It ain’t the bricks it’s the plan — take II

A recent review in Neuron (vol. 88 pp. 681 – 677 ’15) gives a possible new explanation of how our brains came to be so different from apes (if not our behavior of late).

You’ve all heard that our proteins are only 2% different than the chimp, so we are 98% chimpanzee. The facts are correct, the interpretation wrong. We are far more than the protein ‘bricks’ that make us up, and two current papers in Cell [ vol. 163 pp. 24 – 26, 66 – 83 ’15 ] essentially prove this.

This is like saying Monticello and Independence Hall are just the same because they’re both made out of bricks. One could chemically identify Monticello bricks as coming from the Virginia piedmont, and Independence Hall bricks coming from the red clay of New Jersey, but the real difference between the buildings is the plan.

It’s not the proteins, but where and when and how much of them are made. The control for this (plan if you will) lies outside the genes for the proteins themselves, in the rest of the genome (remember only 2% of the genome codes for the amino acids making up our 20,000 or so protein genes). The control elements have as much right to be called genes, as the parts of the genome coding for amino acids. Granted, it’s easier to study genes coding for proteins, because we’ve identified them and know so much about them. It’s like the drunk looking for his keys under the lamppost because that’s where the light is.

We are far more than the protein ‘bricks’ that make us up, and two current papers in Cell [ vol. 163 pp. 24 – 26, 66 – 83 ’15 ] essentially prove this.

All the molecular biology you need to understand what follows is in the following post —

The neuron paper is detailed and fascinating to a neurologist, but toward the end it begins to fry far bigger fish.

Until about 10 years ago, molecular biology was incredibly protein-centric. Consider the following terms — nonsense codon, noncoding DNA, junk DNA. All are pejorative and arose from the view that all the genome does is code for protein. Nonsense codon means one of the 3 termination codons, which tells the ribosome to stop making protein. Noncoding DNA means not coding for protein (with the implication that DNA not coding for protein isn’t coding for anything).

Well all that has changed. The ENCODE Consortium showed that well over half (and probably all) our DNA is transcribed into RNA — for details see This takes energy, and it is doubtful (to me at least) that organisms would waste this much energy if the products were not doing something useful.

I’ve discussed microRNAs elsewhere — for details please see — They don’t code for protein either, but control how much of a given protein is made.

The Neuron paper concerns lncRNAs (long nonCoding RNAs). They don’t code for protein either and contain over 200 nucleotides. There are a lot of them (10,000 – 50,000 are known to be expressed in man. Amazingly 40% of them are expressed in the brain, and not just in adult life, but during embryonic development. Expression of some of them is restricted to specific brain areas. It is easier for an embryologist to tell what type a cell is during brain cortical development by looking at the lncRNAs expressed than by the proteins a given cell is making. The paper contains multiple examples of the lncRNAs controlling when and where a protein is made in the brain.

lncRNAs can contain multiple domains, each of which has a different affinity for a particular RNA (such as the mRNA for a protein), or DNA, or protein. In the nucleus they influence the DNA binding sites of transcription factors, RNA polymerase II, the polycomb repressor complex. The review goes on with many specific examples of lncRNA function — synaptic plasticity, neurotic extension.

Getting back to proteins, the vast majority are nearly the same in all mammals (this is where the 2% Chimpanzee argument comes from). Here is where it gets interesting. Roughly 1/3 of lncRNAs found in man are primate specific. This includes hundreds of lncRNAs found only in man. The paper gives evidence that hundreds of them have shown evidence of positive selection in humans.

So the paper provides yet another mechanism (with far more detail than I’ve been able to provide here) for why our brains are so much larger, and different in many ways than our nearest evolutionary ancestor, the chimpanzee. This is the largest molecular biological difference found so far for the human brain as opposed to every other brain. Fascinating stuff. Stay tuned. I think this is a watershed paper.

Man’s best friend

I usually pay little attention to animal models of neurologic disease. After all, our brain is what separates us from animals (recent human behavior excepted). Neuromuscular disease is different because our peripheral nerves and muscles work the same way as animals. An astounding paper from Harvard and Brazil, gives us an entirely new angle to treat muscular dystrophy, particularly the Duchenne form. I ran a muscular dystrophy clinic for 15 years in the 70s and 80s and haplessly watched young boys deteriorate and die from Duchenne. The major therapeutic advance during that time was — hold your breath — lighter weight braces, allowing the boys to stay out of wheelchairs a bit longer.

Some background for those who don’t know, the molecular defect in Duchenne was found in ’87. Interestingly Kunkel, one of the authors on the original paper [ Cell vol. 51 pp.; 919 – 928 ’87 ] is an author on the present one [ Cell vol. 163 pp. 1204 – 1213 ’15 ]. Duchenne dystrophy affects only males, as the gene for the protein (dystrophin) is found on the X chromosome, so women with a normal X and a mutant X escape. To show how pathetic things were back then, we tried to find out if a sister of a patient was a carrier. How did we do it. By measuring an enzyme released by damaged muscle (CPK) on several occasion. Carriers often showed an elevation.

The mutated protein is called dystrophin. It hooks the contractile apparatus of a muscle cell to the membrane. Failure of this makes muscle cells more fragile when they contract resulting in eventual loss. From a molecular biological point of view the protein is fascinating. The gene is one of largest known, stretching over 2,220,233 positions (nucleotides) on the X chromosome and containing 79 exons. Figuring a transcription rate of 100 nucleotides a second, it takes 6 hours to make the messenger RNA (mRNA) for it. The protein has 3,685 amino acids and figuring a translation rate of 3 – 6 amino acids/second it takes 10 minutes for the ribosome to make it. Given that it takes only 3 nucleotides to code for an amino acid, the protein coding part of the gene takes up only .5% of the gene. Correctly splicing out the introns is a huge task, which we all perform well. This size and complexity of the gene explains why mutations are so common, making it the most common form of hereditary muscular dystrophy (most are).

There are currently all sorts of efforts underway to correct the mutation, particularly in a milder form called Becker dystrophy. Derek has covered them and they constitute a logical direct attack on the pathology.

What is so remarkable about the current Cell paper is that it gives us an entirely new and different way to attack Duchenne (and possible all forms of muscular dystrophy). It involves a colony of dogs in Brazil. They have GRMD (Golden Retriever Muscular Dystrophy) with a mutation in one of the many splice sites in dystrophin (it has 79 exons in man) leading to a premature stop codon and no functional dystrophin in the dogs’ muscles. The animals weaken and become non ambulatory with a shortened lifespan. However, a few of the dogs in the colony seemed pretty normal. So they went to work. The obvious reason was that gene was in some way repaired so the animals had normal amounts of dystrophin. Not so, even though ambulatory, the animals’ muscles had no dystrophin. So the whole genome was sequenced. What they found was that a mutation at an upstream site of a protein called Jagged1 lead to increased transcription of the gene and increased levels of the protein.

Jagged1 is a protein ligand for the Notch system of receptors. The Notch system is important in muscle regeneration. The myoblasts of the animals had more proliferative capacity. The Notch system is far too complicated to go into here —, but expect to see a lot more research money pumped into it.

What I find so fabulous about this paper, is that it gives us an entirely new way of thinking about Duchenne, totally unrelated to the genetic defect, which had been our focus up to now. It also rubs our noses in how little we understand about our molecular biology and cell physiology. If we really understood things, we’d have been focused on Notch years ago. Yet another reason drug discovery is so hard. We are trying to alter a system we only dimly understand.


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