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S P E C I A L
F E A T U R E
E d i t o r i a l
The New World of Clinical Genomics
Leslie G. Biesecker
Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of
Health, Bethesda, Maryland 20892
n 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
I
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
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2012 by The Endocrine Society
doi: 10.1210/jc.2012-3288 Received September 5, 2012. Accepted September 14, 2012.
Abbreviation: MPS, Massively parallel sequencing.
For article see page E2140
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jcem.endojournals.org
J Clin Endocrinol Metab, November 2012, 97(11):3912–3914
J Clin Endocrinol Metab, November 2012, 97(11):3912–3914
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
jcem.endojournals.org
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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.
Acknowledgments
Address all correspondence and requests for reprints to: Leslie G.
Biesecker, M.D., Chief, Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of
Health, 49 Convent Drive, Room 4A56, Bethesda, Maryland
20892. E-mail: [email protected].
The author is supported by the intramural research program
of the National Human Genome Research Institute (NHGRI) of
the National Institutes of Health (NIH). The opinions expressed
here are his own and do not necessarily reflect an opinion or
assessment of the NHGRI or NIH.
Disclosure Summary: The author is an uncompensated consultant to the Illumina Corporation and serves in a compensated
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Biesecker
Comment on Clinical Genomic Research
editorial role for the Wiley-Blackwell Corporation (American
Journal of Medical Genetics Part A).
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