[Update April 6, 2014: The initial title of this post was "23andme genotypes are all wrong". While that was and remains a technically correct statement, I have changed it because the readership of my blog, and this post in particular, has changed. Initially, when I made this post, the readers of the blog were (computational) biologists with extensive knowledge of genotyping and association mapping, and they could understand the point I was trying to make with the title. However in the past few months the readership of my blog has grown greatly, and the post is now reaching a wide public audience. The revised title clarifies that the content of this post relates to the point that low error rates in genotyping can be problematic in the context of genome-wide association reports because of multiple-testing.]

I have been reading the flurry of news articles and blog posts written this week about 23andme and the FDA with some interest. In my research talks, I am fond of displaying 23andme results, and have found that people always respond with interest. On the teaching side, I have subsidized 23andme testing for volunteer students in Math127 who were interested in genetics so that they could learn about personalized genomics first-hand. Finally, a number of my former and current students have worked at 23andme, and some are current employees.

Despite lots of opinions being expressed about the 23andme vs. FDA kerfuffle, I believe that two key points have been ignored in the discussions:

  1. All 23andme genotypes that have ever been reported to customers are wrong. This is the case despite very accurate genotyping technology used by 23andme.
  2. The interpretation of 23andme results involves examining a large number of odds ratios. The presence of errors leads to a huge multiple-testing problem.

Together, these issues lead to an interesting conundrum for the company, for customers, and for the FDA.

I always find it useful to think about problems concretely. In the case of 23andme, it means examining actual genotypes. Fortunately, you don’t have to pay the company $99 dollars to get your own- numerous helpful volunteers have posted their 23andme genotypes online. They can be viewed at openSNP.org where “customers of direct-to-customer genetic tests [can] publish their test results, find others with similar genetic variations, learn more about their results, get the latest primary literature on their variations and help scientists find new associations”. There are a total of 624 genotypes available at openSNP, many of them from 23andme. As an example, consider “samantha“, who in addition to providing her 23andme genotype, also provides lots of phenotypic information. Here is the initial part of her genotype file:

# This data file generated by 23andMe at: Wed Jul 20 20:37:11 2011
#
# Below is a text version of your data. Fields are TAB-separated
# Each line corresponds to a single SNP.  For each SNP, we provide its identifier 
# (an rsid or an internal id), its location on the reference human genome, and the 
# genotype call oriented with respect to the plus strand on the human reference 
# sequence.     We are using reference human assembly build 36.  Note that it is possible 
# that data downloaded at different times may be different due to ongoing improvements 
# in our ability to call genotypes. More information about these changes can be found at:
# https://www.23andme.com/you/download/revisions/
# 
# More information on reference human assembly build 36:
# http://www.ncbi.nlm.nih.gov/projects/mapview/map_search.cgi?taxid=9606&build=36
#
# rsid	chromosome	position	genotype
rs4477212	1	72017	AA
rs3094315	1	742429	AG
rs3131972	1	742584	AG
rs12124819	1	766409	AA
rs11240777	1	788822	AA
rs6681049	1	789870	CC
rs4970383	1	828418	CC
rs4475691	1	836671	CC
rs7537756	1	844113	AA
rs13302982	1	851671	GG
rs1110052	1	863421	GT
...

Anyone who has been genotyped by 23andme can get this file for themselves from the website (by clicking on their name, then on “Browse Raw Data” from the pull-down menu, and then clicking on “Download” in the top-right corner of the browser window). The SNPs are labeled with rsid labels (e.g. rs3094315) and correspond to specific locations on chromosomes (e.g. chr1:742429). Since every human is diploid, two bases are shown for every SNP; one came from mom and one from dad. The 23andme genotype is not phased, which means that you can’t tell in the case of rs3094315 whether the A was from mom and the G from dad, or vice versa (it turns out paternal origin can be important, but that is a topic for another post).

A key question the FDA has asked, as it does for any diagnostic test, is whether the SNP calls are accurate. The answer is already out there. First, someone has performed a 23andme replicate experiment precisely to assess the error rate. In an experiment in 2010 with two replicates, 85 SNPs out of about 600,000 were different. Today, Illumina types around 1 million SNPs, so one would expect even more errors. Furthermore, a replicate analysis provides only a lower bound, since systematic errors will not be detected. Another way to examine the error rate is to look at genotypes of siblings. That was written about in this blog post which concluded there were 87 errors. 23andme currently uses the Illumina Omni Express for genotyping, and the Illumina spec sheet claims a similar error rate to those inferred in the blog posts mentioned above. The bottom line is that even though the error rate for any individual SNP call is very very low (<0.01% error), with a million SNPs being called there is (almost) certainly at least one error somewhere in the genotype. In fact, assuming a conservative error rate leading to an average of 100 errors per genotype, the probability that a 23andme genotype has no errors is less than 10^(-40).

The fact that 23andme genotypes are wrong (i.e. at least one error in some SNP) wouldn’t matter if one was only interested in a single SNP. With very high probability, it would be some other SNPs that are the wrong ones. But the way people use 23andme is not to look at a single SNP of interest, but rather to scan the results from all SNPs to find out whether there is some genetic variant with large (negative) effect. The good news is that there isn’t much information available for the majority of the 1 million SNPs being tested. But there are, nevertheless, lots of SNPs (thousands) to look at. Whereas a comprehensive exam at a doctor’s office might currently constitute a handful of tests– a dozen or a few dozen at most– a 23andme test assessing thousands of SNPs and hundreds of diseases/traits constitutes more diagnostic tests on an individual at one time than have previously been performed in a lifetime.

To understand how many tests are being performed in a 23andme experiment, it is helpful to look at the Interpretome website. The website allows a user to examine information on SNPs without paying, and without uploading the data. I took a look at Samantha, and the Interpretome gave information about 2829 SNPs. These are SNPs for which there is a research article that has identified the SNP as significant in some association study (the website conveniently provides direct links to the articles). For example, here are two rows from the phenotype table describing something about Samantha’s genetic predisposition for large head circumference:

Head circumference (infant) 11655470  CC  T  .05    4E-6    22504419
Head circumference (infant) 1042725    CC  T  .07    3E-10  22504419

Samantha’s genotype at the locus is CC, the “risk” allele is T, the odds ratios  are very small (0.05,0.07) and the p-values are apparently significant. Interpretome’s results differ from those of 23andme, but looking at the diversity of phenotypes reported on gives one a sense for the possibilities that currently exist in genetics, and the scope of 23andme’s reports.

From the estimates of error rates provided above, and using the back of an envelope, it stands to reason that about 1/3 of 23andme tested individuals have an error at one of their “interesting” SNPs. Not all of SNPs arising in association studies are related to diseases, but many of them are. I don’t think its unreasonable to postulate that a significant percentage of 23andme customers have some error in a SNP that is medically important. Whether such errors are typically false positives or false negatives is unclear, and the extent to which they may lead to significant odds ratios is another interesting question. In other words, its not good enough to know how frequently warfarin sensitivity is being called incorrectly. The question is how frequently some medically significant result is incorrect.

Of course, the issue of multiple testing as it pertains to interpreting genotypes is probably a secondary issue with 23andme. As many bloggers have pointed out, it is not even clear that many of 23andme’s odds ratios are accurate or meaningful. A major issue, for example, is the population background of an individual examining his/her genotype and how close it is to the population on which the GWAS were performed. Furthermore, there are serious questions about the meaning of the GWAS odds ratios in the case of complex traits. However I think the issue of multiple testing is a deeper one, and a problem that will only be exacerbated as more disease SNPs are identified. Having said that, there are also approaches that could mitigate errors and improve fidelity of the tests. As DECODE genetics has demonstrated, imputation and phasing can in principle be used to infer population haplotypes, which not only are useful for GWAS analyses, but can also be used to identify erroneous SNP calls. 23andme’s problem is that although they have many genotypes, they are from diverse populations that will be harder to impute and phase.

The issue of multiple testing arising in the context of 23andme and the contrast with classic diagnostics reminds me of the dichotomy between whole-genome analysis and classic single gene molecular biology. The way in which customers are looking at their 23andme results is precisely to look for the largest effects, i.e. phenotypes where they appear to have high odds of contracting a disease, or being sensitive to some drug. This is the equivalent of genome scientists picking the “low hanging fruit” out of genome-wide experiments such as those performed in ENCODE. In genomics, scientists have learned (with some exceptions)  how to interpret genome-wide analyses after correcting for multiple-hypothesis testing by controlling for false discovery rate. But are the customers of 23andme doing so? Is the company helping them do it? Should it? Will the FDA require it? Can looking at ones own genotype constitute too much testing?

There are certainly many precedents for superfluous harmful testing in medicine. For example, the American Academy of Family Physicians has concluded that prostate cancer PSA tests and digital rectal exams have marginal benefits that are outweighed by the harm caused by following up on positive results. Similar arguments have been made for mammography screening. I therefore think that there are serious issues to consider about the implications of direct-to-consumer genetic testing and although I support the democratization of genomics, I’m glad the FDA is paying attention.

samantha

Samantha’s type 2 diabetes risk as estimated from her genotype by Interpretome. She appears to have a lower risk than an average person. Does this make it ok for her to have another cookie?