We’ve found the mutation causing your disease — not so fast, says this paper

This post takes a while to get to the main points, but hang in there, the results are striking (and disturbing).

First: a bit of history.  In the bad old days (any time over about 30 years ago) there was basically only one way to look for a disc in the spinal canal pressing on a nerve producing symptoms (usually pain, followed by numbness and weakness).  It was the myelogram, where a spinal tap was done, an oily substance (containing iodine which Xrays don’t penetrate well)  was injected into the spinal canal, and Xrays taken.   The disc showed up as a defect in the column of dye (not really a dye as any chemist can see).  This usually led to surgery if a disc was found, even if it was one or two spinal levels from where clinicians thought it should be based on their examination and other tests such as electromyography (EMG).  This was usually put down to anatomic variability.  Results were less than perfect.

Myelography was a rather stressful procedure, and I usually brought patients into the hospital the night before, got a cardiogram (to make sure their heart could take it, and that they hadn’t had a silent heart attack).  Then the myelography itself, which wasn’t painful as the radiologist put the needle in under fluoroscopy so they could see exactly where to go.  However many people got severe post-spinal headaches (invariably doctor’s wives), sometimes requiring a blood patch to plug the hole where the (large) needle used to inject the ‘dye’ went — it had to be large because the ‘dye’ was rather oily (viscous).  The bottom line was that you didn’t subject a patient to a myelogram unless they were having a significant problem. Only very symptomatic people had the test, and usually when nonsurgical therapy had been tried and failed.

Fast forward to the MRI (Magnetic Resonance Imaging) era (nuclear magnetic resonance to the chemist, but radiologists were smart enough to get the word nuclear removed so patients would submit to the test).  A painless technique, but stressful for some because of the close quarters in the MRI machine.  You could look at the whole spinal canal, and see far more anatomic detail, because you actually see the disc (rather than its impression on a column of dye) and the surrounding bones, ligaments etc. etc.

What did we find?  There were tons of people with discs where they shouldn’t be (e.g. herniated discs) who were having no problems at all.  This led to a lot more careful assessment of patients, with far better correlation of anatomic defect and clinical symptoms.

What in the world does this all have to do with the genetics of disease?  Patience;  you’re about to find out.

There’s an interesting interview with Eric Lander (of Human Genome Project fame) in the current PNAS (p. 11319).  He notes that in 1990 sequencing a single genome cost $3,000,000,000.  He thinks that at some time in the next 5 years we’ll be able to do this for $1,000, a 3 million-fold improvement in cost.  The genome has around 3,000,000,000 positions to sequence.  As things stand now, it’s literally nothing to determine the sequence of a few million positions in DNA.

On to Cell vol. 145 pp. 1036 – 1048 ’11 which sequenced some 9,000,000 positions of DNA.  This didn’t make a big splash (but its implications might). Just a single paper, buried in the middle of the 24 June ’11 Cell — it didn’t even rate an editorial. Now, as chemists, if you’re a bit shaky on what follows, all the background you need can be found in the series of articles found here –https://luysii.wordpress.com/category/molecular-biology-survival-guide/

As a neurologist, I treated a lot of patients with epilepsy (recurrent convulsions, recurrent seizures).  2% of children and 1% of adults have it (meaning that half of the kids with it will outgrow it, as did the wife of an old friend I saw this afternoon).  Some forms of epilepsy run in families with strict inheritance (like sickle cell anemia or cystic fibrosis). 20 such forms have been tied down to single nucleotide polymorphisms (SNPs)  in 20 different genes coding for protein  (there are other kinds of genes) — all is explained in the background material above).  17/20 of these SNPs are in a type of protein known as an ion channel.  These channels are present in all  our cells, but in neurons they are responsible for the maintenance of a membrane potential across the membrane, which has the ability change abruptly causing an nerve cell to fire an impulse.  In a very simplistic way, one can regard a convulsion (epileptic seizure) as nerve cells gone wild, firing impulses without cease, until the exhausted neurons shut down and the seizure ends.

However, the known strictly hereditary forms of epilepsy account for at most 1 – 2% of all people with epilepsy.  The 9,000,000 determinations of DNA sequence were performed on 237 ion channel genes, but just those parts of the genes actually coding for amino acids (these are the exons).   They studied 152 people with nonhereditary epilepsy (also known as idiopathic epilepsy) and, most importantly, they looked at the same channels in 139 healthy normal people with no epilepsy at all.

Looking at the 17/237  ion channels known to cause strictly hereditary epilepsy they found that 96% of cases of nonhereditary (idiopathic) epilepsy had one or more missense mutations (an amino acid at a given position different than the one that should be there).  Amazingly, 70% of normal people also had missense mutations in the 17.  Looking at the broader picture of all 237 channels, they found 300 different mutations in the 139 normals, of which 23 were in the 17.  Overall they found 989 SNPs in all the channels in the whole group, of which 415 were nonsynonumous.

Well what about mutational load?  Suppose you have more than one mutation in the 17 genes.  77% the cases with idiopathic epilepsy had 2 or more mutations in the 17, but so did 30% of the people without epilepsy at all.

The relation between myelography and early genetic work on disease should be clear.  Back then, a lot was taken as abnormal as only the severely afflicted could be studied, due to time, money and technological constraints.  As the authors note “causality cannot be assigned to any particular variant”.  Many potentially pathogenic genetic variants in known dominant channel genes are present in normals.

What was not clear to me from reading the paper is whether any of the previously described mutations in the 17  are thought to be causative of strictly hereditary epilepsy were present in the 139 normals.

A very interesting point is how genetically diverse the human population actually is (and they only studied Caucasians and Hispanics — apparently no Blacks).  No individual was free of SNPs.  No two individuals (in the 139 + 152) had the same set of SNPs.  Since they found 989 SNPs in the combined group, even in this small sample of proteins (17 of 20,000) this averages out to more than 3 per individual.  Well, are there ‘good’ SNPs in the asymptomatic group, and ‘bad’ SNPs in the patients with idiopathic epilepsy?  Not really, the majority of the SNPs were present in both groups.

I leave it to your imagination what this means for ‘personalized medicine’.   We’re literally just beginning to find out what’s out there.  This is the genetic analog of the asymptomatic disc.  We may not know all we thought we knew about genetics and disease. Heisenberg must be smiling, wherever he is.

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  • Bryan  On July 17, 2011 at 7:31 pm

    It would have been useful if the researchers had characterized the error rate of their PCR + sanger sequencing approach (both steps can introduce errors in the sequence determination) to measure how many of their observed SNP are in fact real, and how many result from errors in the sequencing process. They claim finding ~11,000 SNPs in the 9 million base pairs they read. A quick web search suggests the accuracy of large scale sequencing efforts to be around one error per 10,000 base pairs read. This suggests that perhaps up to 10% of the SNPs they “discovered” are actually sequencing errors. Perhaps this level of error doesn’t affect the conclusions of the paper too much, but it would have been nice if the authors had addressed this point (especially for publication in Cell).

    -Bryan (aka Yggdrasil)

  • luysii  On July 17, 2011 at 8:00 pm

    Bryan: Good point. But assuming the distribution of ‘real’ SNPs in the idiopathic epilepsy group and the normal controls remains the same (I can see no reason why a systemic error rate should change it), their point stands, and quite a point it is.

  • Curious Wavefunction  On July 17, 2011 at 8:32 pm

    One of the tragedies about the hype regarding personalized medicine is that it ignores the fact that personalized medicine is already here. For instance, certain cancer drugs can definitely be tailored toward people who overexpress both p53 and MDM2. Similarly we know that the alkylating anticancer drug temozolomide is not effective in patients who express the MGMT methyl transferase that gets rid of the donated methyl group. Personalized medicine is already here, the problem is that we really need to look at the actual molecular level events and not just correlations from genomics to truly validate it.

    • luysii  On July 18, 2011 at 11:34 am

      [ Nature vol. 475 pp. 101 – 105 ’11 ] Whole genome sequencing of 4 cases of chronic lymphocytic leukemia (CLL) found a mere 46 somatic mutations (and this just in protein coding genes). Then 363 other patients with CLL were studied focusing on the 46 genes found in the first set. It found that 4 of these genes were recurrently mutated in the larger group — NOTCH1, EXPORTIN1 (XPO1) myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases with mutated immunoglobulin genes, while NOTCH1 and XPO1 are found mainly in paitnets with unmutated imunoglobulins. However the recurrence frequency of the 4 genes only ranged from 12% to 2%. This is hardly a plug for the likelihood of personalized medicine. It also shows just how hard the problem of cancer really is, and the genetic heterogeneity underlying what docs have called the same disease (CLL).

      Now that the technology has given us the ability to look, a Pandora’s box has been opened. Better to know, but there’s a huge amount of work ahead.

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