Protein mutation — the view from the bedside — and the lab

Watching a little kid writhe in pain from a Sickle cell crisis hitting his bones, is enough to impress any medical student with how little a protein has to change to cause trouble.  This was the mid-sixties and Sickle Cell anemia was one of the few hereditary diseases whose mutation was actually known.  It is just Valine instead of Glutamic acid at amino acid #6 of the beta-hemoglobin gene.  The rest of the 146 amino acids are unchanged.  Even though it is now 60 years and counting since Pauling found its basis, we have no treatment based on manipulating the 3 dimensional structure of beta globin as it is found in the hemoglobin tetramer. For details see

But now that we can sequence genomes, we know that “for the most densely studied genes, such as globin genes, it appears that virtually every codon in the gene has had a corresponding disease causing mutation observed” [ Neuron vol. 68 p. 245 ’10 ].  That’s not to say that all 146 * 19 = 2,831 possible amino acid changes in beta globin have been found to cause disease, but it’s still impressive.

This isn’t an isolated finding.  Consider CFTR, the protein mutated in cystic fibrosis (the most common genetic disease of Caucasians).  It  contains 1460 amino acids.  Skipping just 1 of them (phenylalanine #507) causes 90% of it.  But by 2003 over 600 mutations in the protein were known (Wikipedia presently has the count at over 1000).

One last example — familial amyloidotic polyneuropathy.  This is a mutation in the transthyretin protein, which is basically in the form of a beta barrel, inside of which snuggles a lipophilic compound) which transthyretin transfers about in the blood stream.  It contains 127 amino acids, and the disease is caused by aggregation of the protein forming amyloid (the similar graveyard of proteins found in the senile plaque of Alzheimer’s disease, although different proteins are involved here).  9 years ago 80 mutations were known, which pretty much covers most of the protein.

Whatever causes the aggregation, it can take a long time to begin.  I saw an 80 woman for essential tremor (without being able to help her very much).  I noted absent reflexes in her legs.  At 85 she developed shooting pains in her legs, and biopsy showed amyloidotic polyneuropathy.  Her brother was also symptom-free until 85+.

So what sort of mindset does this produce in the physician?  Just that proteins are delicate things and that you don’t have to change very much to produce serious problems. Note that all diseases mentioned involve changes in the 3 dimensional structure of the linear protein chain (e.g. their tertiary structure).  There are lots of other diseases, involving enzymes usually, where tertiary structure is preserved, but an amino acid is changed at the active site.

Sickle cell disease is even more interesting, because beta globin is well able to do its job of schlepping oxygen around.  It’s only when certain conditions arise that it loses its tertiary structure  and aggregates inside red blood cells making their shape jagged and them less pliable so that instead of bending as they flow through capillaries (which are no larger and sometimes smaller than the red cell), they get stuck causing the crisis.

So there are more constraints on proteins other than their tertiary structure.  It must be stable enough to resist significant tertiary structure changes under all sorts of physiologic conditions (and pathologic ones as well).

But all of the above is about changes in the amino acids in proteins which cause trouble.  Motoo Kimura has argued that most mutations are selectively neutral.  We will know the answer to this question (probably within a year).  Why?

Just to show have fast things are moving along — Nature vol. 467 pp. 1026 – 1027 ’10 asked 90 genomics centers and labs around the world to estimate the number of sequenced human genomes (not just individual genes from them but the whole 3.2 megaBase genome) they will have in hand by the end of 2011.  The total was 30,000.  This will be only 11 years after the first genome was sequenced.  Amazing.  We’ll certainly be in position to estimate just how many silent amino acid changes there are in proteins in the 30,000 (silent in the sense of not causing disease) and just how fault tolerant the proteins making us up really are.

It’s going to be truly fascinating.  To whet your interest, check out Nature vol. 468 pp. 1050 – 1061 ’10 which is a report of 3 pilot studies of the 1000 Genome project (which will eventually have 2500 genomes).  Apparently proteins are quite fault tolerant, as are we as entire organisms.  They state that on average each person carries 250 – 300 loss of function variants in annotated (protein coding) genes, and 50 – 100 variants previously implicated (which is not to say proven) in inherited disorders.  They also estimate that each of us differs from the reference human genome sequence at 10,000 nonsynoymous sites (e.g. different amino acids) in protein coding genes.

They found a lot of really serious gene defects in the 1000 genomes they studied.  For what the following terms mean see and the next one.

Each of us probably has 200 in frame deletions in protein genes (we only have 20,000 or so such genes), 100 premature stop codons, 50 splice site disruptions, and 250 frameshift mutations.  It’s truly amazing that any of us are alive, if we’re so full of mistakes in our genes.  If true, it shows that humans are even more fault tolerant than their proteins.

So there’s evidence both ways.  Docs see the havoc that mutations can cause (did I mention that I ran a muscular dystrophy clinic, most of which is inherited), while the genome sequencing boys see lots of genomic trouble, in (presumably) normal people walking around.

Stay tuned, it’s really going to be fascinating.

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