Category Archives: Molecular Biology

Junk that isn’t

The more we understand, the more we realize how little we’ve understood what we thought we understood.   Here is a double example.

We have 1,400,000 Alu elements in our genome.  They are about 300 nucleotides long, meaning that there is over 1 every 3,000 nucleotides in our 3,200,000,000 nucleotide genome.  They don’t code for protein, and were widely thought to be junk, selfish genes whose only role was to ensure that the organism carrying them, kept them along as they reproduced.

This post contains a heavy dose of contemporary molecular biology.  If you’re a little shaky on some of it have a look at — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ — and follow the links.

Not so says Proc. Natl. Acad. Sci. vol. 117 pp. 415 – 425  ’20.  They are part of several important physiologic processes (1) T lymphocyte activation (2) heat shock stress (3) endoplasmic reticulum stress.  All 3 cause transcription of Alu’s by RNA polymerase III (pol III).

All RNA levels increase with heat shock, including RNAs made from Alu elements.  They bind directly and tightly (nanoMolar affinity) to RNA polymerase II (which transcribes protein coding genes) and co-occupy the promoters of repressed genes, preventing transcription of these genes and protein synthesis of them.  At least that was the state of play 11 years ago (PNAS 105 5569 – 5574 ’09)

This paper notes that Alu is not passive, but actually a self-cleaving ribozyme (an enzyme made of RNA), an entirely new role.  When complexed with another protein EZH2 (a polycomb protein thought to be a transcriptional repressor using its lysine methylation activity), the rate of Alu self-cleavage increases by 40%.

So what?

In addition to stoping transcription, Alu also retards transcription elongation.  So stress increases in EZH2 causes Alu to cleave itself faster, turning off  repression and improving the responses to the 3 types of stresses above.

So we really didn’t understand both Alu which has been studied for years, or EZH2 a polycomb protein (ditto).  Alu is a self-cleaving ribozyme, and EZH2 doesn’t just turn off genes by its enzymatic activity (lysine trimethylation), but binds to an RNA so it can cleave itself faster (e.g. its a cofactor).

Fascinating and humbling to see how much there is to know about things we thought we knew.  But it’s also exciting.  Who knows what else is out there to discover about the known, never mind the known unknowns.

How little we really understand about proteins

How little we really understand about proteins.  We ‘know’ that the 7 transmembrane alpha helices of G Protein Coupled Receptors (GPCRs) all contain hydrophobic amino acids, so they dissolve in the (hydrophobic) lipids of the membrane.  GPCRs have been intensively by chemists, molecular biologists, pharmacologists and drug chemists with the net result that as of last year “128 GPCRs are targets for drugs listed in the Food and Drug Administration Orange Book. We estimate that ∼700 approved drugs target GPCRs, implying that approximately 35% of approved drugs target GPCRs.” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820538/

So if you changed the hydrophobic amino acids found in the 7 transmembrane segments of GPCRs to hydrophilic ones — all hell should break loose.

Wrong says Proc. Natl. Acad. Sci. vol. 116 pp. 25668 – 25667 ’19 ].  The trick was to replace hydrophobic amino acids with hydrophilic ones with the same shape.

Thus leucine (L — single amino acid letter code) is replaced by glutamine (Q), Isoleucine (I) and Valine (V) is replaced by Threonine (T) and finally phenylalanine (F) is replaced by Tyrosine (Y).  They call this the QTY code.

Instead of destroying the structure of the GPCRs (CCR5 and CXCR4) they became water soluble, and bound their ligands CCL5 for CCR5  and CXCL12 for CXCR4 to the same extent.

Even more amazing, the QTYdesigned receptors exhibit remarkable thermostability in the presence of arginine and retained ligand-binding activity after heat treatment at 60 °C for 4 h and 24 h, and at 100 °C for 10 min.

I would never have expected this.  Would you?

Why did they even do it?  Because GPCR structures are hard to study. You either have to remove them en bloc from the membrane or dissolve them in other lipids so they don’t denature.  Why these two GPCR’s?    Because their ligands are proteins and can’t snuggle deep down inside the 7 alpha helices embedded in the membrane (they’re just too big), but bind to the outside surface.  CCL5 is an 8 kiloDalton protein (probably 80 amino acids, while CXCL12 has 93.  So just solublizing the GPCR without changing any of the amino acids external to the membrane, produces an object for study.

It would be amusing to do the same thing for a GPCR binding one of the monamines.  I doubt that they would bind, but I never would have believed this possible in the first place.

Now is the winter of our discontent

One of the problems with being over 80 is that you watch your friends get sick.  In the past month, one classmate developed ALS and another has cardiac amyloidosis complete with implantable defibrillator.  The 40 year old daughter of a friend who we watched since infancy has serious breast cancer and is undergoing surgery radiation and chemo.  While I don’t have survivor’s guilt (yet), it isn’t fun.

Reading and thinking about molecular biology has been a form of psychotherapy for me (for why, see the reprint of an old post on this point at the end).

Consider ALS (Amyotrophic Lateral Sclerosis, Lou Gehrig disease).  What needs explaining is not why my classmate got it, but why we all don’t have it.  As you know human neurons don’t replace themselves (forget the work in animals — it doesn’t apply to us).  Just think what the neurons  which die in ALS have to do.  They have to send a single axon several feet (not nanoMeters, microMeters, milliMeters — but the better part of a meter) from their cell bodies in the spinal cord to the muscle the innervate (which could be in your foot).

Supplying the end of the axon with proteins and other molecules by simple diffusion would never work.  So molecular highways (called microtubules) inside the axon are constructed, along which trucks (molecular motors such as kinesin and dynein) drag cargos of proteins, and mRNAs to make more proteins.

We know a lot about microtubules, and Cell vol. 179 pp. 909 – 922 ’19 gives incredible detail about them (even better with lots of great pictures).  Start with the basic building block — the tubulin heterodimer — about 40 Angstroms wide and 80 Angstroms high.  The repeating unit of the microtubule is 960 Angstroms long, so 12 heterodimers are lined up end to end in each repeating unit — this is the protofilament of the microtubule, and our microtubules have 13 of them, so that’s 156 heterodimers per microtubule repeat length which is 960 Angstroms or 96 nanoMeters (96 billionths of a meter).  So a microtubule (or a bunch of microtubules extending a meter has 10^7 such repeats or about 1 billion heterodimers.  But the axon of a motor neuron has a bunch of microtubules in it (between 10 and 100), so the motor neuron firing to  the muscle moving my finger has probably made billions and billions of heterodimers.  Moreover it’s been doing this for 80 plus years.

This is why, what needs explaining is not ALS, but why we don’t all have it.

Here’s the old post

The Solace of Molecular Biology

Neurology is fascinating because it deals with illnesses affecting what makes us human. Unfortunately for nearly all of my medical career in neurology ’62 – ’00 neurologic therapy was lousy and death was no stranger. In a coverage group with 4 other neurologists taking weekend call (we covered our own practices during the week) about 1/4 of the patients seen on call weekend #1 had died by on call weekend #2 five weeks later.

Most of the deaths were in the elderly with strokes, tumors, cancer etc, but not all. I also ran a muscular dystrophy clinic and one of the hardest cases I saw was an infant with Werdnig Hoffman disease — similar to what Steven Hawking has, but much, much faster — she died at 1 year. Initially, I found the suffering of such patients and their families impossible to accept or understand, particularly when they affected the young, or even young adults in the graduate student age.

As noted earlier, I started med school in ’62, a time when the genetic code was first being cracked, and with the background then that many of you have presently understanding molecular biology as it was being unravelled wasn’t difficult. Usually when you know something you tend to regard it as simple or unimpressive. Not so the cell and life. The more you know, the more impressive it becomes.

Think of the 3.2 gigaBases of DNA in each cell. At 3 or so Angstroms aromatic ring thickness — this comes out to a meter or so stretched out — but it isn’t, rather compressed so it fits into a nucleus 5 – 10 millionths of a meter in diameter. Then since DNA is a helix with one complete turn every 10 bases, the genome in each cell contains 320,000,000 twists which must be unwound to copy it into RNA. The machinery which copies it into messenger RNA (RNA polymerase II) is huge — but the fun doesn’t stop there — in the eukaryotic cell to turn on a gene at the right time something called the mediator complex must bind to another site in the DNA and the RNA polymerase — the whole mess contains over 100 proteins and has a molecular mass of over 2 megaDaltons (with our friend carbon containing only 12 Daltons). This monster must somehow find and unwind just the right stretch of DNA in the extremely cramped confines of the nucleus. That’s just transcription of DNA into RNA. Translation of the messenger RNA (mRNA) into protein involves another monster — the ribosome. Most of our mRNA must be processed lopping out irrelevant pieces before it gets out to the cytoplasm — this calls for the spliceosome — a complex of over 100 proteins plus some RNAs — a completely different molecular machine with a mass in the megaDaltons. There’s tons more that we know now, equally complex.

So what.

Gradually I came to realize that what needs explaining is not the poor child dying of Werdnig Hoffman disease but that we exist at all and for fairly prolonged periods of time and in relatively good shape (like my father who was actively engaged in the law and a mortgage operation until 6 months before his death at age100). Such is the solace of molecular biology. It ain’t much, but it’s all I’ve got (the religious have a lot more). You guys have the chemical background and the intellectual horsepower to understand molecular biology — and even perhaps to extend it.

 

Barking up the wrong therapeutic tree in Alzheimer’s disease

Billions have been spent by big pharma (and lost) trying to get rid of the senile plaque of Alzheimer’s disease.  The assumption has always been that the plaque is the smoking gun killing neurons.  But it’s just an assumption which a recent paper has turned on its ear [ Proc. Natl. Acad. Sci. vol. 116 23040 – 23049 ’19 ]

It involves a protein, likely to be a new face even to Alzheimer’s cognoscenti.  The protein is called SERF1A (in man) and MOAG-4 in yeast. It enhances amyloid formation, the major component of the senile plaque.  SERF1A is clearly doing something important as it has changed little from the humble single yeast cell to man.

The major component of the senile plaque is the aBeta peptide of 40 and/or 42 amino acids.  It polymerizes to form the amyloid of the plaque.  The initial step of amyloid formation is the hardest, getting a bunch of Abeta peptides into the right conformation (e.g. the nucleus) so others can latch on to it and form the amyloid fiber.   It is likely that the monomers and oligomers of Abeta are what is killing neurons, not the plaques, otherwise why would natural selection/evolution keep SERF1A around?

So, billions of dollars later, getting rid of the senile plaque turns out to be a bad idea. What we want to do is increase SERF1A activity, to sop up the monomers and oligomers. It is a 180 degree shift in our thinking. That’s the executive summary, now for the fascinating chemistry involved.

First the structure of SERF1A — that is to say its amino acid sequence.  (For the nonChemists — proteins are linear string of amino acids, just as a word is a linear string of characters — the order is quite important — just as united and untied mean two very different things). There are only 68 amino acids in SERF1A of which 14 are lysine 9 are arginine 5 Glutamic acid and 5 Aspartic acid.  That’s interesting in itself, as we have 20 different amino acids, and if they occurred randomly you’d expect about 3 -4 of each.  The mathematicians among you should enjoy figuring out just how improbable this compared to random assortment. So just four amino acids account for 33 of the 68 in SERF1A  Even more interesting is the fact that all 4 are charged at body pH — lysine and arginine are positively charged because their nitrogen picks up protons, and glutamic and aspartic acid are negatively charged  giving up the proton.

This means that positive and negative can bind to each other (something energetically quite favorable).  How many ways are there for the 10 acids to bind to the 23 bases?  Just 23 x 22 x 21 X 20 X 19 X 18 x 17 x 16 x 15 x 14 or roughly 20^10 ways.  This means that SERF1A doesn’t have a single structure, but many of them.  It is basically a disordered protein.

The paper shows exactly this, that several conformations of SERF1 are seen in solution, and that it binds to Abeta forming a ‘fuzzy complex’, in which the number of Abetas and SERF1s are not fixed — e.g. there is no fixed stoichiometry — something chemists are going to have to learn to deal with — see — https://luysii.wordpress.com/2018/12/16/bye-bye-stoichiometry/.  Also different conformations of SERF1A are present in the fuzzy complex, explaining why it has such a peculiar amino acid composition.  Clever no?  Let’s hear it for the blind watchmaker or whatever you want to call it.

The paper shows that SERF1 increases the rate at which Abeta forms the nucleus of the amyloid fiber.  It does not help the fiber grow.  This means that the fiber is good and the monomers and oligomers are bad.  Not only that but SERF1 has exactly the same effect with alpha-synuclein, the main protein of the Lewy body of Parkinsonism.

So the paper represents a huge paradigm shift in our understanding of the cause of at least 2 bad neurological diseases.   Even more importantly, the paper suggests a completely new way to attack them.

Technology marches on — or does it?

Technology marches on — perhaps.  But it certainly did in the following Alzheimer’s research [ Neuron vol. 104 pp. 256 – 270 ’19 ] .  The work used (1) CRISPR (2) iPSCs (3) transcriptomics (4) translatomics to study Alzheimer’s.  Almost none of this would have been possible 10 years ago.

Presently over 200 mutations are known in (1) the amyloid precursor protein — APP (2) presenilin1 (3) presenilin2.  The presenilins are components of the gamma secretase complex which cleaves APP on the way to the way to the major components of the senile plaque, Abeta40 and Abeta42.

There’s a lot of nomenclature, so here’s a brief review.  The amyloid precursor protein (APP) comes in 3 isoforms containing 770, 751 and 695 amino acids.  APP is embedded in the plasma membrane with most of the amino acids extracellular.  The crucial enzyme for breaking APP down is gamma secretase, which cleaves APP inside the membrane.  Gamma secretase is made of 4 proteins, 2 of which are the presenilins.  Cleavage results in a small carboxy terminal fragment (which the paper calls beta-CTF) and a large amino terminal fragment. If beta secretase (another enzyme) cleaves the amino terminal fragment Abeta40 and Abeta42 are formed.  If alpha secretase (a third enzyme) cleaves the amino terminal fragment — Abeta42 is not formed.   Got all that?

Where do CRISPR and iPSCs come in?  iPSC stands for induced pluripotent stem cells, which can be made from cells in your skin (but not easily).  Subsequently adding the appropriate witches brew can cause them to differentiate into a variety of cells — cortical neurons in this case.

CRISPR was then used to introduce mutations characteristic of familial Alzheimer’s disease into either APP or presenilin1.  Some 16 cell lines each containing a different familial Alzheimer disease mutation were formed.

Then the iPSCs were differentiated into cortical neurons, and the mRNAs (transcriptomics) and proteins made from them (translatomics) were studied.

Certainly a technological tour de force.

What did they find?  Well for the APP and the presenilin1 mutations had effects on Abeta peptide production (but they differered).  Both however increased the accumulation of beta-CTF.  This could be ‘rescued’ by inhibition of beta-secretase — but unfortunately clinical trials have not shown beta-secretase inhibitors to be helpful.

What did increased beta-CTF actually do — there was enlargement of early endosomes in all the cell lines.   How this produces Alzheimer’s disease is anyone’s guess.

Also quite interesting, is the fact that translatomics and transcriptomics of all 16 cell lines showed ‘dysregulation’ of genes which have been associated with Alzheimer’s disease risk — these include APOE, CLU and SORL1.

Certainly a masterpiece of technological virtuosity.

So technology gives us bigger and better results

Or does it?

There was a very interesting paper on the effect of sleep on cerebrospinal fluid and blood flow in the brain [ Science vol. 366 pp. 372 – 373 ’19 ] It contained the following statement –”

During slow wave sleep, the cerebral blood flow is reduced by 25%, which lowers cerebral blood volume  by ~10%.  The reference for this statement was work done in 1991.

I thought this was a bit outre, so I wrote one of the authors.

Dr. X “Isn’t there something more current (and presumably more accurate) than reference #3 on cerebral blood flow in sleep?  If there isn’t, the work should be repeated”

I got the following back “The old studies are very precise, more precise than current studies.”

Go figure.

Why don’t serotonin neurons die like dopamine neurons do in Parkinson’s disease

Say what ?  “This proportion will likely be higher in rat dopaminergic neurons, which have even larger axonal arbors with ~500,000 presynapses, or in human serotonergic neurons, which are estimated to extend axons for 350 meters” – from [ Science vol. 366 3aaw9997 p. 4 ’19 ]

I thought I was reasonably well informed but I found these numbers astounding, so I looked up the papers.  Here is how such statement can be made with chapter and verse.

“The validity of the single-cell axon length measurements for dopaminergic and cholinergic neurons can be independently checked with calculations based on the total volume of the target territory, the density of the particular type of axon (axon length per volume of target territory), and the number of neuronal cell bodies giving rise to that type of axonThese population analyses are made possible by the availability of antibodies that localize to different types of axons: anti-ChAT for cholinergic axons (also visualized with acetylcholine esterase histochemistry), anti-tyrosine hydroxylase for striatal dopaminergic axons, and anti-serotonin for serotonergic axons.

The human data for axon density and neuron counts have been published for forebrain cholinergic neurons and for serotonergic neurons projecting from the dorsal raphe nucleus to the cortex, and cortical volume estimates for humans are available from MRI analyses; forebrain cholinergic neuron data is also available for chimpanzees. These calculations lead to axon length estimates of 107 m and 31 m, respectively, for human and chimpanzee forebrain cholinergic neurons, and an axon length estimate of 170–348 meters for human serotonergic neurons.”

H. Wu, J. Williams, J. Nathans, Complete morphologies of basal forebrain cholinergic neurons in the mouse. eLife 3, e02444 (2014). doi: 10.7554/eLife.02444; pmid: 24894464

How in the world can these neurons survive as long as they do?

Not all of them do–  At birth there are 450,000 neurons in the substantia nigra (one side or both sides?), declining to 275 by age 60.  Patients with Parkinsonism all had cell counts below 140,000 [  Ann. Neurol. vol. 24 pp. 574 – 576 ’88 ]. Catecholamines such as dopamine and norepinephrine are easily oxidized to quinones, and this may be the ‘black stuff’ in the substantia nigra (which is latin for black stuff).

Here are the numbers for serotonin neurons in the few brain nuclei (dorsal raphe nucleus) in which they are found.  Less than dopamine.  A mere 165,000 +/- 34,000 — https://www.ncbi.nlm.nih.gov › pubmed

So being too small to be seen with a total axon length of a football field, they appear to last as long as we do.  Have we missed a neurological disease due to loss of serotonin neurons?

Why should the axons of dopamine, serotonin and norepinephrine neurons be so long and branch so widely?  Because they release their transmitters diffusely in the brain, and diffusion is too slow, so the axonal apparatus must get it there and release it locally into the brain’s extracellular space, no postsynaptic specializations are present in volume neurotransmission — that’s the point.  This is one of the reasons that a wiring diagram of the brain isn’t enough — https://luysii.wordpress.com/2011/04/10/would-a-wiring-diagram-of-the-brain-help-you-understand-it/.

Just think of that dopamine neuron with 500,000 presynapses.  Synthesis and release must be general, as the neuron couldn’t possibly address an individual synapse.

The more we know the more remarkable the brain becomes.

 

Neurons synapsing with tumor cells, unbelievable but true

As a neurologist, I’ve seen more than enough breast cancer metastatic to the brain.  I never, in a million years, would have though that brain neurons would be forming synapses with them, helping them grow in the process.  But that’s exactly what two papers in the current Nature prove [ Nature vol. 573 pp. 499 – 501, 526 – 531 ’19 ]

The evidence is pretty good.  There are electron micrographs of brain metastases showing breast cancer cells acting like glia, surrounding a synapse between two neurons.  There are synaptic vesicles right next to the presynaptic membrane of the neuron which is apposed to the postsynaptic neuron (that’s what a synapse is after all). They are also present in the same neuron, whose membrane is tightly apposed  to a tumor cell, which stains positive for a type of glutamic acid receptor (the NMDAR).

Breast cancer types have been subdivided by the proteins they contain and don’t contain.  A particularly nasty one, is called triple negative — lacking the estrogen receptor the progesterone receptor and the herceptin receptor. Triple negative breast cancers account for 15 – 20% of all breast cancers, and some 40% of this group will die of brain metastases.  This paper may explain why.

The paper did some work using immunodeficient mice, transplanting human triple negative breast cancer cells into the brain.  Synapses formed between the mouse neurons and the breast cancer cells.

It is known that NMDAR signaling promotes growth tumor growth in other cancer types, and that increased NMDAR expression in breast cancer cells is associated with poor prognosis.

It is incredible to think that the brain is forming synapses with metastatic tumor cells to help them grow, but that’s what must be faced.

The excellent study confined itself to breast cancer metastatic to brain, but the study of other tumors (particularly lung) is sure to follow.

Hell of a way for a cell to defend itself

Imagine it’s Dodge City in the 1880’s and rustlers and gunslingers are shooting up the town.  What do Wyatt Earp and Bat Masterson do?  They don’t call in the cavalry.  They open the jail.   With the town in flames the army moves in, destroying much of the town in the process.

Crazy?  Yes.  But this is a pretty good analogy to how retroviruses are used to help fight off infection if Proc. Natl. Acad. Sci. vol. 116 pp. 17399 – 17408 ’19 is correct

Endogenous retroviruses constitute around 10% of our genome — that’s 320,000,000 nucleotides. Normally they are kept locked up in a tightly condensed mess of DNA and proteins called heterochromatin, so their genome can’t be transcribed and cause trouble.  It takes a lot to lock them up — TRIM28 acting in concert with zinc finger proteins, SUMO, a histone methyltransferase (SETDB1) and last but not least NuRD (not NeRD) — Nucleosome Remodeling and Deacetylation complex.

But when a variety of cells are infected with a variety of influenza viruses, they are let loose because the heterochromatin breaks up, the endogenous retroviruses are freed producing lots of double stranded RNA.  This is sensed as nonSelf, by Pattern Recognition Receptors (PRRs) strongly activating antiviral immunity.

The key thing in the repressive complex is SUMO, a protein resembling ubiquitin which gloms onto lysines like ubiquitin.  To be efficient in repressing the retroviruses, TRIM28 must be SUMOylated.  In some way influenza virus infection results in nonSUMOylated TRIM28.  The authors note that the mechanism of the switch in SUMOylation status ‘has yet to be precisely defined’.  I’m sure the authors are working on it, but who in the world would have thought that cells would use retroviruses as a defense mechanism.  Not me, certainly.

What can dogs tell us about cancer, and (wait for it) sexually transmitted disease

What can 546 dogs tell us about cancer, and STDs (sexually transmitted diseases)?  An enormous amount ! [ Science vol 365 pp. 440 – 441, 464 3aau9923 1 –> 7 ’19 ].  You may have heard about the transmissible tumor that has reduced the Tasmanian Devil population from its appearance in ’96 by 80%.  The animals bite each other transmitting the tumor.  Only 10 – 100 cells are transferred, but death occurs within a year.  The cells survive because Tasmanian devels have low genetic diversity.

The work concerns a much older transmissible tumor (Canine Transmissible Venereal Tumor — aka CTVT) which appeared in Asia an estimated 6,000 year ago, and began dispersing worldwide 2,000 years ago.   Unlike the Tasmanian devil tumor, the tumor is usually cleared by the immune system.

The Science paper has 80+ authors from all over the world, who sequenced the protein coding part of the dog genome (the exome) to a > 100fold depth.   The exome contains 43.6 megabases.   The tumor is transmitted by sex, and the authors note that this mode of transmission nearly requires a rather indolent clinical course, as the animal must survive long enough to transmit the organism again.  This fits with syphilis, AIDs, gonorrhea.  Contrast this with anthrax, cholera, plague which spread differently and kill much faster.

So what does CTVT tell us about cancer?   Quite a bit.  First some background.  The Cancer Genome Atlas (CGA) was criticized as being a boondoggle, but it at least gave us an idea of how many mutations are present in various cancers– around 100 in colon and breast cancers.

Viewed across all dogs, the CTVT genome is riddled with somatic mutations (as compared to the genome of the dog carrying the tumor) –148,030 single nucleotide variants (3.4/1000 !) 12,177 insertion/deletions.  Of the 20,000 dog genes only 2,000 didn’t contain a mutation.   This implies that most genes in the mammalian genome aren’t needed by the cancer cells.  The CTVTs also show no signs of the high rates of chromosomal instability seen in human tumors.

The work provides evidence that cancer isn’t inherently progressive.  This gives hope that some relatively indolent human cancers (say cancer of the prostate) can be controlled.  This calls for ‘adaptive therapy’  — something that limits tumor  growth rather than trying to kill every cancer cell with curative therapy which, if it fails, essentially selects for more aggressive cancer cells.

Some 14,412 genes have 1 mutation changing the amino acid sequence (nonSynonymous) and 5,704 have protein truncating mutations.  The ratio of synonymous to non synonymous mutations is about 3 implying that the mutations which have arisen haven’t been selected for (after all the triplet code for 20 amino acids and 1 stop codon has 64 possibilities), so the average amino acid has 3 codons for it.  This is called neutral genetic drift.

They also found 5 mutated genes present in all 541 tumors — these are the driver mutations, 3 are well known, MYC, PTEN, and retinoblastoma1.

Tons to think about here.  I’ll be away for a few weeks traveling and playing music, but this work should keep you busy thinking about its implications.

 

 

Schizophrenia research, the good news and the bad news

If you are an identical twin whose twin is schizophrenic, your chances of getting schizophrenia is 40%, if you are just a fraternal twin your chance is 15%, amazingly much higher than the 1% chance the rest of us  have, because that’s the incidence in the general population. For what schizophrenia is really like see the old post after the *** at the end

So to find out what causes schizophrenia, study the genes of schizophrenics and compare them to those without it.     [ Neuron vol. 103 pp. 203 – 216 ’19 ] First off —  the Psychiatric Genomics Consortium (PGC) has identified well over 100 (genomic loci) loci with a significant genome-wide association with risk for schizophrenia.  This means that unlike cystic fibrosis (where over 1,700 disease associated mutations have been found in the causative gene),and despite the genetics schizophrenia is not going to be due to one gene.

The good news is that serious money and attention to the genomes of schizophrenics is being paid.The paper reports the latest results from the horribly named BrainSeq.  This is a precompetitive initiative launched by the Liber Institute for Brain Development (LIBD) with heavy big pharma involvement (Eli Lilly, Johnson and Johnson, Hoffman-LaRoche, AstraZenica).    The LIBD has over 1,900 human  postmortem neuropsychiatric disease and control samples.   They are mapping all sorts of genetic information (DNA sequences, RNA sequencing, DNA epigenetics (cytosine methylation) etc. etc.)

The bad news is what this research is telling us.  The paper looked in differences in messenger RNA (mRNA) levels in two areas of the brain in 286 schizophrenics and 265 normal controls.  mRNA levels are a marker for gene expression, levels of the proteins coded for by the mRNA would be better, but is presently beyond our technology (when you are looking at the whole genome, as they were).

Well, out of our 20,000 or so protein coding genes, they found 48 differently expressed (by schizophrenics compared to normals) in one area (the hippocampus) and 245 in another (the dorsolateral prefrontal cortex).  That’s not a big deal, the two areas of the brain have rather different neurons and organization.

The bad news is there was almost no overlap between the 48 and the 245.  So although schizophrenics express their genome differently than normals, the expression varies in brain areas.  It would be great if there was some overlap, so then the genes differentiating schizophrenics from normal could be intensively studied.

The work also casts a shadow over a lot of earlier work, in which gene expression in schizophrenic brain was studied either in one area (or in ground up whole brain), and the results were assumed to be applicable to the brain as a whole.  They aren’t.  Back to the drawing board.

*****

What is schizophrenia really like ?

The recent tragic death of John Nash and his wife warrants reposting the following written 11 October 2009

“I feel that writing to you there I am writing to the source of a ray of light from within a pit of semi-darkness. It is a strange place where you live, where administration is heaped upon administration, and all tremble with fear or abhorrence (in spite of pious phrases) at symptoms of actual non-local thinking. Up the river, slightly better, but still very strange in a certain area with which we are both familiar. And yet, to see this strangeness, the viewer must be strange.”

“I observed the local Romans show a considerable interest in getting into telephone booths and talking on the telephone and one of their favorite words was pronto. So it’s like ping-pong, pinging back again the bell pinged to me.”

Could you paraphrase this? Neither can I, and when, as a neurologist I had occasion to see schizophrenics, the only way to capture their speech was to transcribe it verbatim. It can’t be paraphrased, because it makes no sense, even though it’s reasonably gramatical.

What is a neurologist doing seeing schizophrenics? That’s for shrinks isn’t it? Sometimes in the early stages, the symptoms suggest something neurological. Epilepsy for example. One lady with funny spells was sent to me with her husband. Family history is important in just about all neurological disorders, particularly epilepsy. I asked if anyone in her family had epilepsy. She thought her nephew might have it. Her husband looked puzzled and asked her why. She said she thought so because they had the same birthday.

It’s time for a little history. The board which certifies neurologists, is called the American Board of Psychiatry and Neurology. This is not an accident as the two fields are joined at the hip. Freud himself started out as a neurologist, wrote papers on cerebral palsy, and studied with a great neurologist of the time, Charcot at la Salpetriere in Paris. 6 months of my 3 year residency were spent in Psychiatry, just as psychiatrists spend time learning neurology (and are tested on it when they take their Boards).

Once a month, a psychiatrist friend and I would go to lunch, discussing cases that were neither psychiatric nor neurologic but a mixture of both. We never lacked for new material.

Mental illness is scary as hell. Society deals with it the same way that kids deal with their fears, by romanticizing it, making it somehow more human and less horrible in the process. My kids were always talking about good monsters and bad monsters when they were little. Look at Sesame street. There are some fairly horrible looking characters on it which turn out actually to be pretty nice. Adults have books like “One flew over the Cuckoo’s nest” etc. etc.

The first quote above is from a letter John Nash wrote to Norbert Weiner in 1959. All this, and much much more, can be found in “A Beatiful Mind” by Sylvia Nasar. It is absolutely the best description of schizophrenia I’ve ever come across. No, I haven’t seen the movie, but there’s no way it can be more accurate than the book.

Unfortunately, the book is about a mathematician, which immediately turns off 95% of the populace. But that is exactly its strength. Nash became ill much later than most schizophrenics — around 30 when he had already done great work. So people saved what he wrote, and could describe what went on decades later. Even better, the mathematicians had no theoretical axe to grind (Freudian or otherwise). So there’s no ego, id, superego or penis envy in the book, just page after page of description from well over 100 people interviewed for the book, who just talked about what they saw. The description of Nash at his sickest covers 120 pages or so in the middle of the book. It’s extremely depressing reading, but you’ll never find a better description of what schizophrenia is actually like — e.g. (p. 242) She recalled that “he kept shifting from station to station. We thought he was just being pesky. But he thought that they were broadcasting messages to him. The things he did were mad, but we didn’t really know it.”

Because of his previous mathematical achievments, people saved what he wrote — the second quote above being from a letter written in 1971 and kept by the recipient for decades, the first quote from a letter written in 12 years before that.

There are a few heartening aspects of the book. His wife Alicia is a true saint, and stood by him and tried to help as best she could. The mathematicians also come off very well, in their attempts to shelter him and to get him treatment (they even took up a collection for this at one point).

I was also very pleased to see rather sympathetic portraits of the docs who took care of him. No 20/20 hindsight is to be found. They are described as doing the best for him that they could given the limited knowledge (and therapies) of the time. This is the way medicine has been and always will be practiced — we never really know enough about the diseases we’re treating, and the therapies are almost never optimal. We just try to do our best with what we know and what we have.

I actually ran into Nash shortly after the book came out. The Princeton University Store had a fabulous collection of math books back then — several hundred at least, most of them over $50, so it was a great place to browse, which I did whenever I was in the area. Afterwards, I stopped in a coffee shop in Nassau Square and there he was, carrying a large disheveled bunch of papers with what appeared to be scribbling on them. I couldn’t bring myself to speak to him. He had the eyes of a hunted animal.