Leading Edge
                Essay
                Distilling Pathophysiology
                from Complex Disease Genetics
                                           1,                      2                           3
                Aravinda Chakravarti, * Andrew G. Clark, and Vamsi K. Mootha
                1
                 Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
                2
                 Cornell University, Ithaca, NY 14850, USA
                3
                 Massachusetts General Hospital, Boston, MA 02114, USA
                *Correspondence: aravinda@jhmi.edu
                 http://dx.doi.org/10.1016/j.cell.2013.09.001
                Technologies for genome-wide sequence interrogation have dramatically improved our ability to
                identify loci associated with complexhumandisease.However,achasmremainsbetweencorrela-
                tionsandcausalitythatstems,inpart,fromalimitingtheoreticalframeworkderivedfromMendelian
                geneticsandanincompleteunderstandingofdiseasephysiology.Hereweproposeasetofcriteria,
                akin to Koch’s postulates for infectious disease, for assigning causality between genetic variants
                andhumandiseasephenotypes.
                    .Thus it is easy to prove that the wearing of tall hats and the carrying of                     incorrect knowledge is worse than no
                    umbrellas enlarges the chest, prolongs life, and confers comparative immunity                   knowledge at all (Brown and Goldstein,
                    from disease; for the statistics show that the classes which use these articles                 1992).
                    are bigger, healthier, and live longer than the class which never dreams of pos-                  Consider that two types of genomic
                    sessing such things. It does not take much perspicacity to see that what really                 surveys, one horizontal and the other ver-
                    makesthisdifferenceisnotthetallhatandtheumbrella,butthewealthandnour-                           tical, are now routine for attempting to
                    ishment of which they are evidence, and that a gold watch or membership of a                    understand human biology and disease.
                    club in Pall Mall might be proved in the same way to have the like sovereign                    In horizontal or broad surveys, we can
                    virtues..                                                                                       obtain the full genome sequence in tens
                                       George Bernard Shaw, The Doctor’s Dilemma (Preface), 1909                    to hundreds of thousands of individuals
                                                                                                                    to sort out which genomic segments are
                                                                                                                    important    and which are innocent
                Distinguishing correlation from causality         der to promote the role of one or more            bystanders, to a particular comparison
                is the essence of experimental science.           genes as being ‘‘causal,’’ rather than just       between individuals, such as those with
                Nowhere is the need for this distinction          ‘‘associated,’’   in  a disease process           versus without coronary artery disease
                greatertodaythanincomplexdiseasege-               (Brown and Goldstein, 1992; Falkow,               or cases with early versus late onset of
                netics, where proof that specific genes            1988, 2004)(Box 1).                               dementia. In contrast, in vertical or deep
                have causal effects on human disease                Below we discuss the nature of the              surveys, we examine the effects of the
                phenotypesremainsanenormousburden                 ‘‘proof’’ that we desire in order to make         genomeastheDNAinformationgetspro-
                andchallenge.Giventhepotentialscienti-            fundamental discoveries in human path-            cessed, and its encoded functions get
                fic and medical payoffs of disease gene            ophysiology. We admit at the outset               executed through its transcriptome, pro-
                discovery (Chakravarti, 2001), we argue           that the answers are not straightforward,         teome, and effectors such as the metab-
                in this Essay of the need for a rigorous ex-      and that there are serious technical              olome. Both of these classes of studies
                amination of the assumptions under                and intellectual impediments to demon-            are relevant to analysis of a disease of
                which we connect genes to phenotypes.             strating causality for the common com-            unknown etiology and have re-empha-
                This is particularly so in this age of routine    plex disorders of man where multiple              sized the long-held suspicion that study-
                -omic surveys, which can produce more             interacting   genes are involved. We              ing genes one-at-a-time may not be
                false-positive than true-positive findings         acknowledge that even unproven candi-             meaningful because a gene’s effect is
                (Kohane et al., 2006). Moreover, genomic          date genes may lead to significant                 usually pleiotropic, context dependent,
                mapping and sequencing approaches                 insight   into  disease pathophysiology.          and contingent upon the state of many
                that are invaluable for producing a list of       Nevertheless, the casual conflation of             other genetic and nongenetic factors
                unbiased candidates are, by themselves,           ‘‘mapped locus’’ to ‘‘proven gene’’ is a          (Chin et al., 2012). In turn, this implies
                insufficientforimplicatingspecificgene(s)           constant     source    of   confusion     and     that proving a gene’s specific role in a
                in a diseaseorbiologicalprocess.Conse-            obfuscation in biology and medicine               biological process, either in wild-type or
                quently, we suggest that specific genetic          that requires remedy. We hope to offer            mutant form, may not be straightforward
                criteria, analogous to Koch’s postulates          some concrete suggestions, however                because its role may only be evident
                in microbiology,needtobesatisfiedinor-             difficult they may be to satisfy, because          when examined in relation to its
                                                                                                         Cell 155, September 26, 2013 ª2013 Elsevier Inc. 21
                  Box1.Koch’sPostulatesforComplex              any overt disease phenotype, presum-             The case of amyotrophic lateral scle-
                  HumanDiseasesandTraits                       ably due to the buffering by other genes       rosis (ALS), a devastating, progressive
                                                               (MacArthur et al., 2012). Acknowledging        motor neuron disease, illustrates this
                      (1) Candidate gene variants are          this complexity, there are two general         point (Ludolph et al., 2012). Despite the
                         enriched in patients.                 ways forward. First, at this stage of our      lack of evidence, we largely describe
                      (2) Disruptionofthegeneinamodel          knowledge, perhaps we should not worry         ALS as being ‘‘heterogeneous’’ and
                         system gives rise to a model          about ‘‘all’’ of the genes in a disease, in    comprised of single-gene mutations that
                         phenotype that is accepted as         many ways an undefinable goal, but              can individually lead to disease. In
                         relevant and ‘‘equivalent’’ to the    rather those whose effects are demon-          1993, mutations in superoxide dismutase
                         humanphenotype.                       strable, i.e., through a mutation that, irre-  1(SOD1) were identified in an auto-
                      (3) The   model    phenotype     can     spective of its interactions, can by itself    somal-dominant form of the disease;
                         be rescued with the wild-type         affect a critical pathway. Second, as we       subsequently, the disorder has become
                         humanalleles.                         unravel the effects of multiple genes on       synonymous with aberrant clearance of
                      (4) Themodelphenotypecannotbe            a phenotype, we should advance the             free radicals as its central pathology.
                         rescued with the mutant human         same criterion, namely, that a set of          What is often not appreciated, however,
                         alleles.                              mutations affects that same critical pro-      is that fewer than 10% of all cases of
                                                               cess. Both of these goals are approach-        ALS are familial and even fewer follow
                                                               able, particularly with recent advances        an apparent Mendelian pattern. Even
                 biochemical partners, and in particular       in   genome-editing     technologies   that    within this subset of cases, more than
                 contexts of diet, pathogen exposure,          allow the creation of multiple mutations       20 distinct genes, spanning other path-
                 etc. (Zerba et al., 1996). This is a partic-  within a single experimental organism          ways including RNA homeostasis, have
                 ular problem in genetic studies of any        (Wang et al., 2013). The question then is      been identified, and SOD1 represents a
                 outbred     nonexperimental      organism,    how ‘‘complex’’ are complex traits and         minority of cases. The molecular etiology
                 suchasthehuman,andstudiesofhuman              diseases?                                      for the majority of the sporadic forms of
                 disease, where investigations are obser-                                                     the disease remains unclear, and the sci-
                 vational not experimental. It is the strong   TheNewGenetics:Understanding                   entific problem in understanding ALS is
                 belief of contemporary human geneticists      the Function of Variation                      more than simply identification of addi-
                 that uncovering the genetic underpin-         With the rediscovery of Mendel’s rules of      tional genes. We may ask, can SOD1
                 nings of any disease, however complex,        transmission more than 100 years ago,          and the other described gene mutations
                 is the surest unbiased route to under-        there was a vicious debate on the              lead to ALS by themselves? Are these
                 standing its pathophysiology and, thus,       relative importance of single-gene versus      the key rate-limiting steps to ALS or sim-
                 enabling its future rational therapies        multifactorial inheritance (Provine, 1971).    ply one of several required in concert? Is
                 (Brooke et al., 2008). Consequently, for      Geneticists quickly, and successfully,         the aberrant clearance of free radicals
                 this view to prevail, we should require       focused on deciphering the specific             the fundamental defect or one of many
                 experimental evidence, be it in cells, tis-   mechanisms of gene inheritance and un-         such pathologies or a common down-
                 sues, experimental models, or the rare        derstanding the physiology of the gene         stream consequence? Given the diver-
                 patient, for the role of a specific gene in    in lieu of answering why some pheno-           sity and number of deleterious, even
                 a disease process. We discuss here the        typeshadcomplexetiologyandtransmis-            loss-of-function, genetic variants in all
                 types of evidence that we consider            sion. Nevertheless, the rare examples of       of our genomes (Abecasis et al., 2012;
                 incontrovertible.                             deciphering the genetic basis of complex       MacArthur et al., 2012) and, in the
                   Successinthisdifficulttaskrequiresus         phenotypes,suchasfortruncate(wing)in           absence of stronger evidence bearing
                 to solve a logical conundrum: how can         Drosophila (Altenburg and Muller, 1920),       on these questions, it is fair to assume
                 we understand the genes underlying a          clearly emphasized that traits were more       that ALS patients harbor multiple muta-
                 phenotype if some of these component          than the additive properties of multiple       tions with a plurality of molecular defects
                 factors, in isolation, do not have recog-     genes. Today, it is quite clear that           and that free radical metabolism is only
                 nizable phenotypes on their own? We           Mendelian inheritance of traits, including     one of a set of canonical pathophysiol-
                 know that even in a simple model organ-       diseases, is the exception not the rule.       ogies that define the disease. No doubt,
                 ism, budding yeast, synthetic lethality—      Nevertheless, the entire language of           this plurality is the case for cancer (Vo-
                 where death or some other phenotype           genetics is in terms of individual genes       gelstein et al., 2013), Crohn’s disease
                 occurs only through the conspiracy of         for  individual   phenotypes, with one         (Jostins et al., 2012), and even rare
                 mutations at two different genes—is           function, rather than the ensemble and         developmental disorders such as Hirsch-
                 widely prevalent (Costanzo et al., 2010).     emergent properties of genomes. This           sprung disease (McCallion et al., 2003).
                 Interactions of greater complexity and        absence of a specific genetics language         In all of these cases, a richer genetics
                 involving more than two genes are also        for the proper description of the multi-       vocabulary may improve our understand-
                 known in yeast (Hartman et al., 2001)         genic architecture of traits (the ensemble)    ing of the phenotypes through recog-
                 and must be true for humans as well. A        remains as an impediment to our under-         nizing what we know and what we
                 human genome will typically harbor 20         standing of the nature and degree of           don’t; our current language limits us to
                 genes that are fully inactivated, without     genetic complexity of the phenotype.           describing genes not phenotypes.
                 22 Cell 155, September 26, 2013 ª2013 Elsevier Inc.
                  Molecular biology, genetics’ twin, on         molecular biology, biochemistry, and             is now applicable to any human trait or
                the other hand, appears to have been far        physiology of the genes within a mapped          disease.    In  fact,   more than 2,000
                more successful in deciphering and              locus to even identify the disease gene,         confirmed loci, each containing multiple
                describing not only its individual compo-       let alone understand its functions. Suc-         genes, affecting susceptibility to more
                nents (e.g., DNA, RNA, protein) but also        cessinthisendeavorwillrequireasynthe-            than 100 medically relevant traits (e.g.,
                their mutual relationships (e.g., DNA-          sis of many biological disciplines that          blood pressure) and disease (e.g., hyper-
                protein interaction) and ensembles (e.g.,       includes the role of genetic variation as        tension) are now known (Hindorff et al.,
                transcriptional complex), although this is      intrinsic to the biological process, not an      2009). For most complextraits examined,
                also far from complete (Watson et al.,          aspect to be ignored.                            many such loci have been mapped, but
                2007). Not only do we understand the               Consequently,meldingvariation-based           the vast majority of the specific genes
                structure of individual genes and how           genetic and molecular biological thinking        remain unidentified. We can sometimes
                their molecular functions get executed,         is of critical importance for both fields         guess at a candidate gene within the lo-
                but we are also starting to learn how           and is central to our understanding of           cus (Jostins et al., 2012), sometimes
                functionsgetregulatedthroughadiversity          mechanisms of trait variation, including         implicate a gene by virtue of an abun-
                of cis- and trans-acting functions. The         interindividual variation in disease risk. If    dance of rare variants among affected
                consequencesoftheprimaryandinterac-             most disease, in most humans, is the             individuals (Jostins et al., 2012), in rare
                tion effects are often well understood,         consequence of the effects of variation          circumstances, use therapeutic modula-
                even though not completely described,           at many genes, then knowledge of their           tion of a pathway to pinpoint the gene
                at both the molecular and cellular levels       functional    relationships,   rather    than    (Moon et al., 2004), and sometimes
                (Alberts et al., 2007). There are also          merely their identities, is central to under-    identify one by painstaking experimental
                improving technologies and understand-          standing the phenotype. This is clearly a        dissection (Musunuru et al., 2010), but,
                ing of the structures and functions of          problem of ‘‘Systems Biology’’ but one           generally, identification of the underlying
                ensembles of proteins and cells, and            that    incorporates     genetic    variation    gene has not become easier. In fact,
                how these interact and communicate              directly. The ability to integrate the real-     mostofthemappedlociunderlyingcom-
                with one another to create complexity           ities of suchwidespreadgeneticvariation,         plex traits remain unresolved at the gene
                (Ilsley et al., 2013). Although the use of      which are ultimately at the causal root          or mechanistic level.
                genetic tools and genetic perspectives          of disease mechanisms, with systems                 Despite the beginning clues to human
                are fundamental to this progress, these         biology approaches to understand func-           disease pathophysiology that complex
                advances have not as yet led to a major         tional contingencies is central to the           disease mapping is providing, and the
                revision of our understanding of trait or       challenge of deciphering complex human           slow identification of individual genes, it
                disease variation. The major reason for         disease. Importantly, it is likely to spur       appears highly unlikely that we can
                this discrepancy is that, with few excep-       newthinking in both fields.                       understand traits and diseases this way.
                tions (Raj et al., 2010), molecular and cell                                                     There is indeed evidence for scenarios
                biology has focused on the impact of            Genetic Dissection of Complex                    in  which variation in complex traits,
                deleting or overexpressing genes and            Phenotypes                                       including risk of complex disease, is
                not grappled with the consequences of           Genetic transmission rules imply that,           mediated by a myriad of variants of
                allelic variation.                              even in an intractable species such as           minute effect, spread evenly across the
                  Classical Mendelian genetics has been         us, one can map genomic segments that            genome (Yang et al., 2011). Therefore,
                a boon to uncovering biology from yeast         must contain a disease or trait gene. The        we need other approaches to override
                to humans whenever a mutation with a            lure and success of this method is that          this bottleneck.
                simple inheritance pattern can be iso-          we can map a disease locus in the                   For Mendelian disorders, gene identifi-
                lated. This approach has been revolution-       absenceofanyknowledgeoftheunderly-               cation within a locus is made possible by
                ary in the unicellular yeast, particularly      ing biology of the phenotype. Such               eachmutationbeingnecessaryandsuffi-
                because genetics (and gene manipula-            mapping requires identification of the            cientforthephenotype,beingfunctionally
                tion), biochemistry, and cell biology were      segregation of common sites of variation         deleterious and rare, and having an inher-
                meldedtounderstandfunctionatavariety            across the genome, now easy to identify          itance pattern consistent with the pheno-
                of levels. This kind of multilevel approach     through sequencing, and recognition of           type. It’s the mutation that eventually
                has been less straightforward, but still        a genomic segment identical-by-descent           reveals the biology and explains the
                largely successful, for a metazoan such         in affected individuals, both within and         phenotype. Any component locus for a
                asDrosophilawheremoregenesandmul-               between families. This task has become           complex disease has no such restriction,
                tiple specialized cells often rescue the        easier and more powerful as sequencing           as the causal variants are neither neces-
                effects of a mutation or enhance its minor      technology has improved to provide a             sary nor sufficient, nor coding (in fact,
                effect. These lessons suggest to us that        nearlycompletecatalogofvariantsabove             theyarefrequentlynoncodingandregula-
                the current approach, based strictly on         1% frequency in the population; further          tory) nor rare (Emison et al., 2010; Jostins
                genetic variation, to understanding com-        improvements to sample rarer variants            etal., 2012).Currently,themajorattempts
                plexhumandiseaseisalsogrosslyinsuffi-            are ongoing (Abecasis et al., 2012).             to overcome this impediment involve reli-
                cient and,asinyeastandflies,willrequire          Consequently, genetic mapping, once              ance on single severe mutations at the
                the contemporaneous analysis of the             the province of rare Mendelian disorders,        very   same component genes and
                                                                                                      Cell 155, September 26, 2013 ª2013 Elsevier Inc. 23
                                                                                                                             complex inheritance          problem (Yosef
                                                                                                                             et al., 2013). Even more importantly, this
                                                                                                                             approach might, through the effect of
                                                                                                                             mutations, allow us to decipher cell cir-
                                                                                                                             cuitry and understand which pathways
                                                                                                                             are limiting and which are redundant.
                                                                                                                             This last aspect is critical: as we argue
                                                                                                                             below, with our current state of knowl-
                                                                                                                             edge, we are likely to have our greatest
                                                                                                                             success with understanding how genes
                                                                                                                             map onto pathways, and how pathways
                                                                                                                             mapontodisease,beforeatruequantita-
                                                                                                                             tive understanding of disease biology
                                                                                                                             emerges.Onemightcounterthatexisting
                                                                                                                             gene ontologies do precisely that, but,
                                                                                                                             even in yeast, this appears to be highly
                                                                                                                             incomplete (Dutkowski et al., 2013).
                                                                                                                             Proving Causality: Molecular
                                                                                                                             Koch’sPostulates
                                                                                                                             The evidence that a specific gene is
                                                                                                                             involved in a particular human disease
                                                                                                                             has historically been nonstatistical and
                                                                                                                             based on our experience with identifying
                   Figure 1. Complementary Approaches Necessary for Proving Genetic Causality and                            mutations in Mendelian diseases. The
                   Understanding the Pathophysiology of Complex Disease                                                      chief criteria have been to demonstrate
                   Geneticassociationstudiesinhumanscansynergizewithpriorknowledgeandsystems-levelquantitative               cosegregation with the phenotype in
                   analysis to generate predictions of what pathways and modules are disrupted, where (anatomically), and    families, exclusivity of the mutation to
                   when(developmentally)toyieldaspecificmorphologicalorbiochemicalphenotype.Thesepredictionscan               affected individuals (rare alleles absent
                   then be tested in an appropriate model system while adhering to the postulates outlined in Box 1.
                                                                                                                             incontrols),andthenatureofthemutation
                                                                                                                             (a   plausibly     deleterious    allele   at   a
                   demonstrating Mendelian inheritance of               ease.Thisapproachhasbeenhighlyprof-                  conserved site within a protein). Unfortu-
                   the same or similar phenotype, and/or                itable in Crohn’s disease—a common                   nately, as already mentioned, all of these
                   identifying single genes with a demon-               inflammatorydisorderwhoserootcauses                   rules break down in complex phenotypes
                   strable excess of rare coding variants.              remained      cryptic    until   genome-wide         where neither cosegregation nor exclu-
                   Thefirstofthesetwostrategiesisastrong                 association studies identified a large                sivity to affecteds nor obviously delete-
                   unproven hypothesis and probably not                 number of loci with fundamental defects              rious alleles are likely; moreover, many
                   universally true, whereas the second re-             in mucosalimmunity(GrahamandXavier,                  mutationsaresuspectedtobenoncoding
                   lies on very large sample sizes of patients          2013)—but not in type 2 diabetes, where              andinadiversityofregulatory RNAmole-
                   and suffers from the unknown functional              the pathophysiology awaits clarification              cules. Consequently, statistical evidence
                   effect of the majority of rare coding vari-          (Groop and Pociot, 2013). Although we                of enrichment has been the mainstay,
                   ants.    Consequently, these strategies              suspect that the numbers of pathways                 but this has two negative consequences:
                   themselvesdependonthehiddenbiology                   involved are fewer than the numbers of               first, scanning across the genome or
                   we seek and are applicable only to the               genes involved, this is merely suspicion.            multiple loci covering tens to hundreds
                   most common human diseases. It ap-                   Nevertheless, can we reduce the com-                 of megabases requires very large sample
                   pears to us that ignorance of biology has            plexity of the problem by identifying all            sizes and very strict levels of significance
                   become rate limiting for understanding               of the relevant pathways? Despite uncer-             toguardagainstthemanyexpectedfalse-
                   diseasepathophysiology,exceptperhaps                 tainty, this approachhastheadvantageof               positive findings; second, genetic effects
                   for the Mendelian disorders. There are               leading to specific testable hypotheses.              that are small or genes with only a few
                   two ways to get out of this vicious cycle            Thesecondapproachistofocusresearch                   causal alleles are notoriously difficult to
                   (Figure 1).                                          onwhythediseaseiscomplexinthefirst                    detect, although they may be very impor-
                      One approach may be to use a set of               place. Although the genome is linear, its            tant to understanding pathogenesis. This
                   model traits and diseases and employ                 expression       and    biology     are    highly    difficulty translates into a low power of
                   their existing mapped loci to identify a             nonlinear and hierarchical, being seques-            detection, as common disease alleles
                   small set of the component genes by                  tered in specific cells and organelles                cannot be distinguished from bystander
                   brute-force (or, luck) and use the uncov-            (Ilsley et al., 2013). Understanding this            associated alleles, whereas rare alleles
                   ered biology to infer which other genes              hierarchy,     the    province     of   systems      are observed too infrequently to provide
                   in their ‘‘pathways’’ can explain the dis-           biology, is critical to the solution of the          statistical    significance.     Consequently,
                   24 Cell 155, September 26, 2013 ª2013 Elsevier Inc.