Targeted Sequencing Bags a Diagnosis

A nice complement to the one paper (Ng et al) I detailed last week is a paper that actually came out just before hand (Choi et al). Whereas the Ng paper used whole exome targeted sequencing to find the mutation for a previously unexplained rare genetic disease, the Choi et al paper used a similar scheme (though with a different choice of targeting platform) to find a known mutation in a patient, thereby diagnosing the patient.

The patient in question has a tightly interlocked pedigree (Figure 2), with two different consanguineous marriages shown. Put another way, this person could trace 3 paths back to one set of great-great-grandparents. Hence, they had quite a bit of DNA which was identical-by-descent, which meant that in these regions any low-frequency variant call could be safely ignored as noise. A separate scan with a SNP chip was used to identify such regions independently of the sequencing.

The patient was a 5 month old male, born prematurely at 30 weeks and with "failure to thrive and dehydration". Two spontaneous abortions and a death of another premature sibling at day 4 also characterized this family; a litany of miserable suffering. Due to imbalances in the standard blood chemistry (which, I wish the reviewers had insisted on further explanation for those of us who don't frequent that world), a kidney defect was suspected but other causes (such as infection) were not excluded.

The exome capture was this time on the Nimblegen platform, followed by Illumina sequenicng. This is not radically different from the Ng paper, which used Agilent capture and Illumina sequencing. At the moment Illumina & Agilent appear to be the only practical options for whole exome-scale capture, though there are many capture schemes published and quite a few available commercially. Lots of variants were found. One that immediately grabbed attention was a novel missense mutation which was homozygous and in a known chloride transporter, SLC26A3. This missense mutation (D652N)targets a position which is almost utterly conserved across the family, and is making a significant change in side chain (acid group to polar non-charged). Most importantly, SLC26A3 has already been shown to cause "congenital chloride-losing diarrhea" (CLD) when mutated in other positions. Clinical follow-up confirmed that fluid loss was through the intestines and not the kidneys.

One of the genetic diseases of the kidney that had been considered was Bartter syndrome, which the more precise blood chemistry did not match. Given that one patient had been suspected of Bartter but instead had CLD, the group screened 39 more patients with Bartter but lacking mutations in 4 different genes linked to this syndrome. 5 of these patients had homozygous mutations in SLC26A3, 2 of which were novel. 190 control chromosomes were also sequenced; none had mutations. 3 of these patients had further follow-up & confirmation of water loss through the gastrointestinal tract.

This study again illustrates the utility of targeted sequencing for clinical diagnosis of difficult cases. While a whole exome scan is currently in the neighborhood of $20K, more focused searches could be run far cheaper. The challenge will be in designing economical panels which will allow scanning the most important genes at low cost and designing such panels well. Presumably one could go through OMIM and find all diseases & syndromes which alter electrolyte levels and known causative gene(s). Such panels might be doable for perhaps as low as $1-5K per sample; too expensive for routine newborn screening but far better than a endless stream of tests. Of course, such panels would miss novel genes or really odd presentations, so follow-up of negative results with whole exome sequencing might be required. With newer sequencing platforms available, the costs for this may plummet to a few hundred dollars per test, which is probably on par with what the current screening of newborns for inborn errors runs. One impediment to commercial development in this field may well be the rapid evolution of platforms; companies may be hesitant that they will bet on a technology that will not last.

Of course, to some degree the distinction between the two papers is artificial. The Ng et al paper actually, as I noted, did diagnose some of their patients with known genetic disease. Similarly, the patients in this study who are now negative for known Bartter syndrome genes and for CLD would be candidates for whole exome sequencing. In the end, what matters is to make the right diagnosis for each patient so that the best treatment or supportive care can be selected.




Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, Nayir A, Bakkaloğlu A, Ozen S, Sanjad S, Nelson-Williams C, Farhi A, Mane S, & Lifton RP (2009). Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proceedings of the National Academy of Sciences of the United States of America, 106 (45), 19096-101 PMID: 19861545

Targeted Sequencing Bags a Rare Disease

Nature Genetics on Friday released the paper from Jay Shendure, Debra Nickerson and colleagues which used targeted sequencing to identify the damaged gene in a rare Mendelian disorder, Miller syndrome. The work had been presented at least in part at recent meetings, but now all of us can digest it in entirety.

The impressive economy of this paper is that they targeted (using Agilent chips) less than 30Mb of the human genome, which is less than 1%. They also worked with very few samples; only about 30 cases of Miller Syndrome have been reported in the literature. While I've expressed some reservations about "exome sequencing", this paper does illustrate why it can be very cost effective and my objections (perhaps not made clear enough before) is more a worry about being too restricted to "exomes" and less about targeting.

Only four affected individuals (two siblings and two individuals unrelated to anyone else in the study) were sequenced, each at around 40X coverage of the targeted regions. Since Miller is so vanishingly rare, the causative mutations should be absent from samples of human diversity such as dbSNP or the HapMap, so these was used as a filter. Non-synonymous (protein-altering), splice site mutations & coding indels were considered as candidates. Both dominant models and recessive models were considered. Combining the data from both siblings, 228 candidate dominant genes and 9 recessive ones fell out. Looking then to the unrelated individuals zeroed in on a single gene, DHODH, under the recessive model (but 8 in the dominant model). Using a conservative statistical model, the odds of finding this by chance were estimated at 1.5x10e-05.

An interesting curve was thrown by nature. If predictions were made as to whether mutations would be damaging, then DHODH was excluded as a candidate gene under a recessive model. Both siblings carried one allele (G605A) predicted to be neutral but another allele predicted to be damaging.

Another interesting curve is a second gene, DNAH5, which was a candidate considering only the siblings' data but ruled out by the other two individuals' data. However, this gene is already known to be linked to a Mendelian disorder. The two siblings had a number of symptoms which do not fit with any other Miller case -- and well fit the symptoms of DNAH5 mutation. So these two individuals have two rare genetic diseases!

Getting back to DHODH, is it the culprit in Miller? Sequencing three further unrelated patients found them all to be compound heterzygotes for mutations predicted to be damaging. So it becomes reasonable to infer that a false prediction of non-damaging was made for G605A. Sequencing of DHODH in parents of the affected individuals confirmed that each was a carrier, ruling out DHODH as a causative gene under a dominant model.

DHODH is known to encode dihydroorotate dehydrogenase, which catalyzes a biochemical step in the de novo synthesis of pyrimidines. This is a pathway targeted in some cancer chemotherapies, with the unfortunate result that some individuals are exposed to these drugs in utero -- and these persons manifest symptoms similar to Miller syndrome. Furthermore, another genetic disease (Nagler) has great overlap in symptoms with Miller -- but sequencing of DHODH in 12 unrelated patients failed to find any coding mutations in DHODH.

The authors point to the possible impact of this approach. They note that there are 7,000 diseases which affect fewer than 200K patients in the U.S. (a widely used definition of rare disease), but in aggregate this is more than 25M persons. Identifying the underlying mutations for a large fraction of these diseases would advance our understanding of human biology greatly, and with a bit of luck some of these mutations will suggest practical therapeutic or dietary approaches which can ameliorate the disease.

Despite the success here, they also underline opportunities for improvement. First, in some cases variant calling was difficult due to poor coverage in repeated regions. Conversely, some copy number variation manifested itself in false positive calls of variation. Second, the SNP databases for filtering will be most useful if they are derived from similar populations; if studying patients with a background poorly represented in dbSNP or HapMap then those databases won't do.

How economical a strategy would this be? Whole exome sequencing on this scale can be purchased for a bit under $20K/individual; to try to do this by Sanger would probably be at least 25X that. So whole exome sequencing of the 4 original individuals would be less than $100K for sequencing (but clearly a bunch more for interpretation, sample collection, etc). The follow-up sequencing would a add a bit, but probably less than one exome's worth of sequencing. Even if a study turned up a lot of candidate variants, smaller scale targeted sequencing can be had for $5K or less per sample. Digging into the methods, the study actually used two passes of array capture -- the second to clean up what wasn't captured well by the first array design & to add newer gene predictions. This is a great opportunity to learn from these projects -- the array designs can keep being refined to provide even coverage across the targeted genes. And, of course, as the cost per base of the sequencing portion continues its downwards slide this will get even more attractive -- or possibly simply be displaced by really cheap whole genome sequencing. If the cost of the exome sequencing can be approximately halved, then perhaps a project similar to this could be run for around $100K.

So, if 700 diseases could each be examined at 100K/disease, that would come out to $70M -- hardly chump change. This underlines the huge utility of getting sequencing costs down another order of magnitude. At $1000/genome, the sequencing costs of the project would stop grossly overshadowing the other key areas - sample collection & data interpretation. If the total cost of such a project could be brought down closer to $20K, then now we're looking at $14M to investigate all described rare genetic disorders. That's not to say it shouldn't be done at $70M or even several times that, but ideally some of the money saved by cheaper sequencing could go to elucidating the biology of the causative alleles such a campaign would unearth, because certainly many of them will be much more enigmatic than DHODH.



Sarah B. Ng, Kati J. Buckingham, Choli Lee, Abigail W. Bigham, Holly K. Tabor, Karin M. Dent, Chad D. Huff, Paul T. Shannon, Ethylin Wang Jabs, Deborah A. Nickerson, Jay Shendure, & Michael J. Bamshad (2009). Exome sequencing identifies the cause of a mendelian disorder Nature genetics : doi:10.1038/ng.499

Sherlock Holmes, Omicist

A nice item in GenomeWeb about a new NIH initiative that's just brilliant -- using omics to try to solve rare disease mysteries. I've blogged on this topic before, and it's an obvious way to go -- particularly since the price of these genome studies is dropping so precipitiously.

As noted by the patient named in the report, finding a cause is not (alas!) the same as finding a treatment. But if many patients with mystery diseases are screened, there will almost certainly be some clues that do lead to useful remedies. It is also important to remember that very rare syndromes often shed important light on very common disorders. For example, a large number of rare tumor syndromes have illuminated key cellular mechanisms broadly relevant to tumorigenesis -- von Hippel-Lindau, neurofibromatosis, and many others. Having some molecular clue to the disease is infinitely better than a baffling list of symptoms.

Sanguine Thoughts

Sometimes in life, you just want to lie back and stare at the ceiling. Other times, you have no choice, which is how I found myself for a while last Sunday morning. I was lying on a simple bed, staring at the ceiling of a high school gymnasium, with tubing coming out of my right arm.

I hate needles. One of the many reasons med school was out for me is that I hate needles. I can eat breakfast while watching a pathology lecture, but I can't stand the sight of a needle going into human skin (nor a scalpel). My fear of needles was so severe I had to be partially sedated once for a blood draw, which was most unfortunate as I then couldn't scream properly when the nurse speared some nerve or another & nobody realized the agony I was in. Sticking myself as an undergraduate didn't help, though at least the needle was fresh and had not yet gone into the mouse.

So, a number of years ago I resolved to fight this irrational fear by confronting it in a positive manner, and so I started to give blood regularly. For a while I was giving pretty much as often was allowable, but in the last few years I've slipped and missed a lot of appointments. But, the Red Cross still calls & I still get in a few times a year. And the needle phobia has been calmed from abject terror to tense dread, a marked improvement. Plus, I feel like I'm doing some good -- your odds of saving someone's life are certainly better for donating than for entering a career in drug discovery (though the latter has some huge tails -- a lucky few get to make an amazing impact)

Most blood drives are held in conference rooms, and so the ceilings aren't terribly interesting. Gym ceilings don't do much for me either. There is one memorable blood drive location I've been to: the Great Hall of the Massachusetts State House. But generally, it's iPod and random thoughts time.

This time, the iPod was giving me the right stuff (as in the soundtrack for the same), but my thoughts were roaming. Having done this a lot, one compares the sensations to previous times. For example, the needle in had a little more burn than usual, perhaps some iodine was riding in? On the way out was even more disconcerting: a warm dripping on my arm! The needle-tubing junction had just failed, but things were rectified quickly (though I looked like an extra from M*A*S*H while I held my arm up).

But most of all, I remembered why I had come to this particular drive, with no thought of letting the appointment slip. This drive was in honor of five local children with Primary Immunodeficiency, and three of them are from a family we know well. Their bodies make insufficient immunoglobulins, leaving the patients vulnerable to various infections. Regular (sometimes as often as weekly) infusions of immunoglobulins are the treatment for this. Some causes of PI are known, but others have yet to be identified.

Given that this family has two unaffected parents and three boys all affected, my mind wanders in some obvious directions. That pattern is most likely due to the mutation lying on the X-chromosome, which sons inherit only from their mother. Given the new advances in targeted sequencing, for a modest amount of money one could go hunting for the mutation on the X -- perhaps a few thousand dollars per patient. Such costs are certainly within the realm of rather modest charity fund-raising, so will we see raffles-for-genomes in the future?

If such efforts are launched, will patients and their families be tempted to go largely on their own, bypassing conventional researchers -- and perhaps conventional ethical review boards? If anyone with a credit card can request targeted sequencing, surely there will be motivated individuals who would do so. Some, like the parent profiled in last week's Nature, will have backgrounds in genetics -- but others probably won't. Let's face it, with a little guidance or a lot of patient reading, the knowledge can be acquired by someone willing to learn the lingo.

As I was on my way out, the middle boy, who is 5, was heading outside with his mother to play. He looked up at me and said sincerely "Thank you for giving me your blood". Wow, did that feel good!

When Personal Genomics is Very Personal

Anyone interested in personal genomics should hunt down the new Nature (available online at the moment) and read the story of Hugh Rienhoff, whose third child (a daughter) was born with a still mysterious set of symptoms. Since her birth he has been bouncing around trying to get a diagnosis for her condition which resembles Marfan's and a similar disorder called Loeys–Dietz.

Rienhoff was trained as a physician under Victor McKusick and helped start a genomics firm (DNA Sciences), so he was a bit primed for this. Remarkably, he has apparently set up his own PCR laboratory in his house so he can perform targeted sequencing of candidate genes from his daughter's DNA -- using an unnamed contract research house. Alas, none of these searches have yet turned anything up.

Because of the similarity of his daughter's symptoms to the other two syndromes & because both of these syndromes involve TGF-beta signalling, as well as the well characterized role of TGF-beta signalling in muscle development & his daughter's muscular problems, Rienhoff & her doctor recently decided to put the child on a high blood pressure medication which is suggested to reduce TGF-beta signalling and to help in a mouse Marfan's model.

The story is a good illustration of the promise -- and the complications -- of cheap DNA sequencing to identify the causes of rare diseases. Small scale targeted sequencing hasn't worked out -- but given the large number of genes known to be involved in TGF-beta signalling the odds were never wonderful. Perhaps a full genome scan, or targeted resequencing using one of the new array-based capture schemes, might find a strong candidate mutation -- some of the other TGF-beta related syndromes are dominants, so perhaps this will be too & comparing the daughter's scan to the parents will single out the mutation. But, the results might be inconclusive -- no strong candidates. Or, perhaps a candidate is found because it is a de-novo mutation in the child & is likely to have a major effect (non-synonymous substitution, truncation mutant, etc), but in an utterly unstudied gene. At least that's something to go on, but not much.

The article touches on how patients with unusual clusters of symptoms often get lumped into 'dustbin' categories, syndromes whose common thread is an inability to assign the patients to another category. Personal genomics may be quite useful for cutting down on such diagnoses, as the genetic data may sometimes provide the compass to guide through the morass of symptoms. On the other hand, there will probably be whole new bins of genetic syndromes -- 'polymorphism in X with skeletal defects' -- again, it is something to go on, but they are almost guaranteed to pile up much faster than the experiments to sort them out can be run.

After reading the article, I can't help but hope that his daughter gets into one of the big sequencing programs, such as the recently announced Venter center 10K genome effort. There will be a lot to be gained by finding out the ordinary variation which makes each one of us different, but there should also be a bunch of slots reserved for patients for whom sequence results might, if they are lucky, give them some new options in life.

A Parent's Worst Nightmare

Today's Globe contained a story sure to cudgel the heart of any parent: an apparently healthy 6-year-old girl collapsed & died during a suburban soccer game this weekend. Details were not yet available, but in such cases one class of causes are cardiac arrythmias.

Such horrible events are very rare, but still very concerning since they injure or kill persons who otherwise would have very long futures ahead of them. One response to this is to suggest screening all young athletes for arrythmias. Like all screening exercises, these run the risk of many false positives, which can incur financial, medical & always emotional costs.

With widespread personal genome sequencing around the corner, there will certainly be interest in trying to use this information to prevent such tragedies. The fact that a number of polymorphisms relevant to such sudden collapses are already known makes this not at all hypothetical. However, just as with screening by other methods, it is likely that such tests would be crude for quite a while going forward -- too many causative mutants will be unknown (false negatives) and some of the seemingly harmful variants will prove to be either incorrectly labeled so or not harmful in the particular personal context (e.g. another variant suppresses the effect). Furthermore, since such events are rare it will be challenging to find more such variants -- especially if there are a large number of rare variants predisposing to such events.

In any case, it's hard not to cross one's fingers -- no parent should have to worry about a routine childhood activity carrying invisible risk.

DNA Under Pressure

Over at Eye on DNA an MD's op-ed column on genetic association studies is getting a good roasting. The naivete about the interplay of genes and the environment suggested by the original article (which would seem to suggest that environment completely trumps genes) reminded me of an idle speculation I've engaged in recently. Now, in this space I frequently engage in speculation, but this involves a celebrity of sorts, which I don't plan to include often. But I think this speculation is interesting enough to share/expose.

The Old Towne Team has been tearing up the American League East this year, much to the glee of the rabid Red Sox fan who lives down the hall from me. A key ingredient to their success is a very strong starting pitching rotation, and leading that rotation in several stats is Josh Beckett, particularly his American League leading record of 9 wins and 0 losses.

Now, as an aside, I have very little respect for baseball statistics. The rules for most seem arbitrary and the number of statistics endless. I have a general suspicion that baseball statisticians believe that some asylum holds a standard deviant, and that Bayes rule has something to do with billiards. But with Beckett on the mound, good things tend to happen.

Beckett was the prize acquisition last year, but at times he looked like a poor buy. The Sox gave up two prospects for him, one of whom hurled a no hitter and the other ended up as NL Rookie of the Year. Last year Beckett had mixed results, but this year he is on fire.

What hath this to do with genetics? Well, for a short time we lost his services due to an avulsion on one of his pitching fingers, which is the technical term for a deep tear in the skin. Beckett has a long history of severe blisters which knock him out of action periodically.

Genes, or the environment? It could be that he just grips the ball in such a way that anyone would lose their skin. But it could also be that he has polymorphisms in some connective tissue genes which make him just a bit more susceptible to this sort of injury. There are many connective tissue disorders known, with perhaps the best known in connection to sports being Marfan's syndrome. Marfan's leads to a tall and lanky physique, ideal for sports such as basketball and volleyball -- and it was Marfan's that killed Olympic star Flo Hyman. Beckett isn't covered with blisters (at least, no such news has reached the press), but what if it is only in the intense pressure of delivering a fastball that the skin gives way. If this were true, the phenotype would be most certainly due to the genotype -- but only in the context of a very specific environmental factor.

Of course, this is a miserable hypothesis to try to test. Perhaps you would scour amateur and professional baseball for pitchers with similar problems and do a case-control study with other pitchers who don't develop blisters. Or, you would need to collect DNA from his relatives, and also teach them all to pitch just like Josh Beckett in order to see if they too develop blisters and avulsions. It could be a first: a genetics study whose consent form includes permission to be entered into the Major League Baseball Draft!

Genome Sequencing for Unique Genetic Diseases

The following is based on a chance encounter with a stranger. I don't believe I am violating any ethical lines, but will entertain criticism in that department. I'm not a physician, and nothing in this should be viewed as more than extreme scientific speculation. If the Gene Sherpa or others feel I should be raked over the coals, then get the bonfire going!

I have a young son & so spend time on playgrounds and similar situations. Last fall I had taken him & his cousin to a playground at a park; he had reached his quota of watching his cousin's youth soccer and needed a change-of-pace. Some older kids were there, resulting in gleeful experimentation with extreme G-forces on the merry-go-round.

One of the other parents there was keeping an eye on the events but also tending to a clearly very challenged girl in a wheelchair -- she had various medical gear on the wheelchair and at least one tube. In the course of routine conversation (no, I wasn't prying!) it came out that (a) the girl was 10 and (b) she had already in her young life had multiple organ transplants. Intrigued, I asked what her condition was called (okay, now I'm guilty of prying), and the answer was that as far as any specialist they had consulted knew, this girl was the only known case.

Given my background & interests, it was natural for me to start the mental wheels grinding on genetic speculation. Don't worry, I don't reserve this for strangers! Shortly after my son was born it came out in casual conversation that a relative on his mother's side was colorblind, and so I went into hyperdrive grinding out the probability that my son would be too. Around the time of his first New Year, he was playing with a green ball amongst red ones, and the lightning hit again! Green amongst red! After several trials, his red-green vision powers were good!

Now, such a multi-symptomatic syndrome could have many causes, but suppose it was genetic? Since there was only one known case, traditional genetic mapping would be impossible. But, what might whole-genome sequencing be able to do?

There are many genetic scenarios, but let's narrow it down to three.

First, it could be a simple Mendelian dominant, as is the case with CHARGE, a developmental disorder. For such devastating diseases to be dominant, they must arise from spontaneous mutations.

Second, it could be a simple Mendelian recessive syndrome, but very rare or a novel phenotype of a known one. Depending on the type of mutation, damaged versions of a gene can lead to phenotypes which are not obviously related. For example, some alleles of decapentaplegic in Drosophila are known as Held Out because the wings are always pointed straight away from the body; other alleles led to the official name as the flies have 15 defective appendages.

Third, it could be something else. Interactions between genes, some nasty epigenetic problem, etc. Those will all be pretty much intractable by genome sequencing.

But how much progress might we make with the other two cases? First, suppose we got a complete genome sequence for the affected child. Comparing that sequence vs. the human reference sequence and catalogs of SNPs (which should grow quite large once human whole genome sequencing becomes common), one could attempt to identify all of the unusual variants in the child's genome. Depending on how well the child's ethnic background is represented in the databases, there might be very few and there might be very many. A sizable deletion or inversion might be regarded as a good candidate for a dominant. An unusual variant, particularly a non-synonymous coding SNP or a SNP in a known or suspected genetic control region, might be a candidate -- and if homozygous might be a candidate for a recessive.

Now, suppose we could also get the mother's sequence as well. Now it should be possible to really hammer on the Mendelian dominant hypothesis, as any rare variants found in the mother can be ruled out, since she is unaffected. If you could get the father's DNA as well, then one could really go to town. In particular, that would enable identifying any de novo mutations (those that occurred in one of the parent's germline (ova/sperm) but they don't carry in their somatic (body) cells). It should also allow identifying any funky transmission issues, such as uniparental disomy (the case in which both copies of a genetic region are inherited from the same parent). Finding uniparental disomy might be a foot in the door towards an imprinting hypothesis -- getting two copies of a gene from the same parent can be trouble if the gene is imprinted.

How many candidate mutations & genes might we find with such a fishing expedition? That is the big question, and one which really can only be answered by trying it out. The precise number of rare alleles found is going to depend on the ethnic background of the parents and their relatedness. For example, if the parents were relatives (consanguineous), then there is a higher chance of getting two copies (homozygosing) of a rare variant (no, I'm not the type to pry that deep). If a parent is from an ethnic background that isn't well represented in the databases, then many rare SNPs will be present.

What sort of gene might we be looking for? Probably just about anything. Genes with known developmental roles might be good candidates, or perhaps predicted transcription factors (ala CHARGE), but it could be anything. Particularly difficult to make sense of would be rare SNPs distant from any known gene -- they might be noise, but they could also affect genetic control elements distant from their target gene, a well-known phenomenon.

For the family in question, what is the probability of getting useful medical information? Alas, probably very slim. One can hope for a House-like epiphany which leads to a treatment, but even if a good candidate for the causative gene can be found that is unlikely. Some genetic diseases involving metabolic enzymes can be managed through diet (e.g. PKU), but many others cannot (e.g. Gaucher's). One might also hope for an enzyme-replacement therapy. However, it is quite likely that such a disease would not be in a metabolic enzyme, and might well be in a gene which we really know nothing much about.

So, such a hunt would be for medical edification. Would it be worth it? At the current price of ~$1M/genome, it's hard to see. But at $10K or $1K per genome, it might well be. It probably wouldn't work in many cases, but perhaps if a program was set up to screen by genome sequencing many families with rare genetic (or potentially genetic) disorders, some successes would filter out. We'd certainly learn a lot of find-scale information about human recombination and de novo mutations.