The new world of clinical genomics.

In 1893, Robert Koch published the now famous postulates laying out the criteria by which a microorganism could be determined to be a cause of disease (1). These postulates have served as practical criteria for this determination of pathogenicity, have been adapted to newer types of data (such as DNA) in infectious diseases (2, 3), and have inspired others to think clearly about the rules for the determination of etiological causation for noninfectious diseases, for example molecular approaches to heritable diseases (4, 5). The first question raised by the publication of “Novel Microcephalic Primordial Dwarfism Disorder Associated with Variants in the Centrosomal Protein Ninein” in this issue of the JCEM (6) is whether they have reached the bar regarding causation for the variant they have identified. The field of human genetics has evolved a general understanding of the criteria for causality of mutations that cause Mendelian disorders. These typically include meiotic analysis (linkage mapping) that localizes the mutations to a genomic region (typically <10 MB, which is about 0.3% of the genome with, on average, 70–80 genes) followed by analysis of multiple unrelated individuals who harbor independent and rare gene variants, coupled with functional data that show that the variants adversely perturb the function of the gene product. I describe this as a “general understanding” for postulates of causation because the criteria are applied a bit more flexibly than are Koch's postulates—different mixes of data can be judged as sufficient to conclude causation. Using these flexible postulates, positional cloning has proven to be a robust method for the determination of the cause of hundreds of Mendelian disorders in the last 25 yr. The advent of massively parallel sequencing (MPS) technologies (7, 8) has upended the positional cloning approach to the identification of the cause of Mendelian disorders. These technologies allow the interrogation of exomes (85–90% of the protein-coding exons in genes) and genomes (or 85–90% of the genome) in a single assay—removing the necessity for meiotic mapping to localize the mutations. This is a huge advance because the meiotic mapping step typically required multiple families with at least two affecteds in each family or a very large extended family with multiple affected individuals to give sufficient linkage information. As demonstrated by Dauber et al. (6), by using MPS, one can identify potential pathogenic sequence variants with a single family with just two affected individuals. However, because MPS does not limit the region of interest as did meiotic mapping, it identifies thousands of candidate gene variants—because the region of interest is, in this case, the entire autosomal portion of the genome. Instead of meiotic mapping, the approach now used is to filter the thousands of sequence variants that are typically identified in MPS experiments. The authors used what little genetic data they had, which was to look for variants shared by the two affected siblings that fit an autosomal recessive model (either homozygous or compound heterozygous). They also filtered the variants by frequency in control genomes, setting a ceiling of 1% allele frequency. This left them with six rare variants. They then used a functional approach to identify which of these genes might be expressed in the growth plate, reasoning that the gene could be expressed there because of the manifestation of extreme short stature in the two affected children. One of those six genes from the MPS was NIN, which was expressed in the growth plate. They went on to characterize NIN, which is known to be involved in centrosomal function (a key pathway in other forms of primordial dwarfism), and to show that there was no obvious defect of centrosomal function in mutant cells. Knocking down the expression of the zebrafish ortholog of NIN led to abnormal brain stem formation and craniofacial anomalies. It is reasonable to ask whether the data presented by Dauber et al. (6), taken together, prove that NIN mutations caused the primordial dwarfism in these two patients. I would suggest that there are two limitations of the data that bear on this question. The functional data are not a perfect recapitulation of the phenotype; ideally, one would like to have seen severe growth reduction in the developing fish. It is unexplained how the expression of NIN in the growth plate is related to the central nervous system and craniofacial defects observed in the knockdown zebrafish. The second relates to the MPS itself—the lack of meiotic localization data combined with the 85–90% coverage of a typical exome or genome sequence means that there may be several hundreds of uninterrogated gene variants. As well, the hypothesis of autosomal recessive inheritance is reasonable, but the possibility that this is an autosomal dominantly inherited trait with incomplete penetrance, markedly reduced expressivity, or mosaicism on one of the parents has not been excluded. The combination of imperfect animal modeling of the trait combined with weak exclusion of alternative hypotheses suggests that the case for causation is not certain. But is it good enough? The authors have apparently concluded that it is not, and I think that is an honest and sober assessment of the data. The authors describe NIN as a candidate gene for microcephalic osteodysplastic primordial dwarfism, not a causative gene. This discovery of NIN as a candidate awaits confirmation in other patients, further animal modeling by knocking out the gene (or knocking in the mutations) in the mouse, and other functional studies. One final point regarding proof of causation should be raised, which is whether the data in this paper are sufficient to warrant publication at this juncture or whether additional data should have been mustered before publication. The arguments for publication are that the disorder is of high interest to the readership; that the experiments are well-designed, properly performed, and thoughtfully interpreted; and that the sum of the evidence is sufficiently strong. As well, the publication of this report may lead to the recognition of NIN mutations in other rare cases of microcephalic osteodysplastic primordial dwarfism that were previously not recognized to be associated with the phenotype. The arguments against are several. If the conclusion is incorrect, it may lead researchers to waste efforts on studying NIN and could lead to clinical misdiagnoses in molecular diagnostics laboratories if the paper is interpreted as conclusive. Also, correcting an erroneous conclusion in the scientific literature is difficult—analogous to how hard it is to get a wrong clinical diagnosis out of a patient's chart. On the other hand, there can be issues even if the conclusion is correct. It may impair the ability of some other hypothetical group of researchers to publish data in a high-impact journal if they have a larger patient cohort and better functional data—they have been “scooped” by spending more time to get better data. Although medical science should not be primarily about publication impact factors, it would be a serious problem for the field if we discouraged publication of thorough investigations, large patient series, and complete stories. One final consideration crosses my mind in reviewing the publication of Dauber et al. (6), which is that of the other 34,604 variants in the MPS data of these siblings. Are there other variants in that dataset that may have relevance to their health or that of their family? This is the issue of the so-called incidental or secondary variants—known pathogenic mutations in genes that are encountered in MPS datasets that are not of primary research interest (9). For example, we have recently demonstrated (10) that among a set of 572 patients subjected to exome sequencing for reasons unrelated to cancer, about 1% of them had rare, apparently highly penetrant mutations in genes that could cause cancer susceptibility. What we also showed was that only half of the patients with these mutations had a suggestive family history of tumors, leading us to conclude that the MPS was the only way this was likely to be detected in these research participants. The ethics of this question are complex, but it is critical to recognize that these data exist and for researchers and Institutional Review Boards to make deliberate and thoughtful choices about whether and how to pursue these variants. But, we are now past the point where researchers can pretend that this issue does not exist, and Institutional Review Boards need to be asking their investigators how they intend to address this challenge and how they are going to explain their answer to the research participants themselves.