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Next-Generation Sequencing Demands Next-GenerationPhenotyping

Raoul C.M. Hennekam1∗ and Leslie G. Biesecker2

1Department of Pediatrics and Translational Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;2Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland

For the Deep Phenotyping Special IssueReceived 27 September 2011; accepted revised manuscript 8 January 2012.Published online 13 February 2012 in Wiley Online Library (www.wiley.com/humanmutation).DOI: 10.1002/humu.22048

ABSTRACT: Next-generation sequencing (NGS) is themost powerful diagnostic tool since the roentgenogram.NGS will facilitate diagnosis on a massive scale, allowinginterrogation of all genes in a single assay. It has been sug-gested that NGS will decrease the need for phenotypingin general and medical geneticists in particular. We arguethat NGS will shift focus and approach of phenotyping.We predict that NGS performed for diagnostic purposeswill yield variants in several genes, and consequences ofthese variants will need to be analyzed and integrated withclinical findings to make a diagnosis. Diagnostic skills ofmedical specialists will shift from a pre-NGS-test differen-tial diagnostic mode to a post-NGS-test diagnostic assess-ment mode. In research phenotyping and medical geneticassessments will remain essential as well. NGS can iden-tify primary causative variants in phenotypes inherited ina Mendelian pattern, but biology is much more complex.Phenotypes are caused by the actions of several genes andepigenetic and environmental influences. Dissecting all in-fluences necessitates ongoing and detailed phenotyping,refinement of clinical diagnostic assignments, and itera-tive analyses of NGS data. We conclude that there will bea critical need for phenotyping and clinical analysis, andthat medical geneticists are uniquely positioned to addressthis need.Hum Mutat 33:884–886, 2012. Published 2012 Wiley Periodi-cals, Inc.∗

KEY WORDS: NGS; whole-exome sequencing; whole-genome sequencing; phenotype; dysmorphology;Mendelian; monogenic

IntroductionNext-generation sequencing (NGS) presents a paradigm shift for

medicine and has the potential to allow more tailored (personal)medical care based on individual risk. As NGS will not only be

∗Correspondence to: Raoul C.M. Hennekam, Department of Paediatrics, Floor H7-

236, Academic Medical Centre, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands.

E-mail: r.c.hennekam@amc.uva.nl

Contract grant sponsor: Intramural Research Program of the National Human Genome

Research Institute of the National Institutes of Health (to L.G.B.).

useful in disorders with a Mendelian pattern of inheritance, butalso in polygenic and multifactorial etiologies, it will become acomponent of medical practice for all disorders, and all medicalspecialties will benefit from these developments. However, it hasbeen suggested that one medical discipline may instead be a victimof this technology: medical genetics, specifically dysmorphology.During a plenary session of the 2011 Meeting of the EuropeanSociety of Human Genetics, an expert and well-respected speakermade this prediction, and one of the topics of a public debate inthe 2011 International Congress of Human Genetics in Montrealwas “. . .medical genetics will disappear as a separate specialty.” Toparaphrase Mark Twain, the rumors of the death of dysmorphologyand medical genetics are greatly exaggerated. So if the rumoreddeath is not imminent, what is afoot?

This rumor of demise reflects not an error of judgment, but a lackof imagination and foresight. We predict that instead of a demise,there will be a transformation—and this transformation will be dra-matic for medical genetic researchers and clinicians, but most im-portantly, for the patients. A main change will be in the approach todiagnosis. Until now, diagnostic molecular tests in clinical medicinehave been low-throughput and expensive. The consequence is thatclinicians spend enormous amounts of time (so money) gatheringdata (medical and family history, physical findings, imaging, etc.),lumping and splitting patients into precise categories, and refiningand debating differential diagnoses to allow them to select a sin-gle test or a small set of tests to determine the most likely clinicaldiagnosis. The diagnostic process has been organized in this waynot because it is intrinsically superior, but because this has beenthe best way to match the throughput of testing to diagnostic real-ities. Now that testing has become high-throughput, clinicians willask whether that approach could be completely re-engineered. Wepredict that re-engineering of the diagnostic process will be drivenby medical geneticists and will create a new, rewarding practice ofmedical genetics, and invigorate, not destroy, the profession.

Medical Genetics Practice in a NGS-DrivenParadigm

Imagine a patient with ataxia. Currently, the clinician performsnumerous tests such as electromyographies, cranial magnetic res-onance imaging, and so on to gather evidence on the most likelytype of ataxia and to help select the gene test that should be ordered.This consumes significant time and healthcare resources. Instead,with NGS, a minimal level of clinical assessment (a concise history,physical examination, and discussion with the patient to obtainpermission to interrogate the genome) will allow the clinician toaccess (of course securely) the genome server in which this patient’s

Published 2012 Wiley Periodicals, Inc. ∗This article is a US Government work and, as such, is in the public domain of the United States of America.

genome sequence is banked.1 With a few keystrokes, the cliniciancan check all genes known to cause ataxia and find the one (orperhaps a few) with a mutation. If there is more than one “hit,”the clinician uses a knowledgebase regarding phenotypes associatedwith mutations to get recommendations on further clinical testinguseful to determine which “hit” is real and allowing identificationof the causative mutation.

Imagine next a preschool-aged patient with an intellectual dis-ability and unusual facial morphology. Currently, the primary carephysician has little to contribute to the diagnostic process and wouldrefer the patient to a medical geneticist. The geneticist would evalu-ate the child for additional malformations, search databases, consultwith experienced colleagues, and start a process of ordering a se-ries of individual tests, with an ultimate diagnostic yield of about50% [Van Karnebeek et al., 2005]. Instead, with NGS, one mightimagine that the primary care physician orders a single blood test(whole-genome sequencing with copy number and structural varia-tion assessment), perhaps combined with a sample from the parents,and will receive an analysis based on an input query of “intellectualdisability and dysmorphic features.”

If the NGS would identify a single, high-penetrance causativemutation, known to be associated with a recognized clinical entity,the family can be counseled by reviewing a knowledgebase of thatdisorder. If instead there were to be multiple candidate causativemutations (a more probable outcome), the primary care clinicianwill refer the family to a medical geneticist or other appropriatespecialist. Further analyses regarding the various possible diagnoseswill be started, based on the genomic hits. The specialists will thenuse their clinical diagnostic skills to distinguish among the candidatemutations, potentially order focused follow-up tests (e.g., imaging),and make a diagnosis. This approach to diagnostics allows specialiststo accelerate the diagnostic process by using the NGS test resultsto focus their evaluations on manifestations, as indicated by theNGS results. The identification of a mutation in a causative genewill facilitate counseling, lead to treatment recommendations, allowaccurate reproductive recurrence risk assessments, and offer familiesthe opportunity for further testing within families and prenatalstudies.

However, it is important to recognize that NGS and computerswill not magically make diagnoses in all, or perhaps even most, pa-tients. If a diagnosis is suggested, that will be wonderful. Typically,however, NGS and informatics will provide a handful of possibil-ities and allow clinicians to perform focused and efficient furtherassessments of the patient. So, clinical skills and acumen of medicalgeneticists will remain essential and only will shift from a pretestdifferential diagnosis generation mode to a post-test diagnostic as-sessment mode. Medical geneticists will use their skills, deployedin a new way, to make more, more accurate and faster diagnoses atlower cost. These potential improvements in the diagnostic processshould engender optimism and excitement among medical geneticsand other specialties as well.

What Do Patients Want to Know?Patients (or parents of young patients) ask simple, direct ques-

tions, and clinicians try to answer them to the best of their abilities.These questions are (1) “what do I have?”; (2) “what causes it?”; (3)

1Note, here we assume in an adult that the sequence was previously generated for

other reasons and was available to be reused for this new purpose (at little marginal

cost). The assumption is that genome interrogation will be inexpensive and likely to be

used early in life (some say for newborn screening), and serve as a life-long resource

to improve the healthcare of an individual.

“what are the consequences for my health?”; and finally, (4) “whatcan be done about it?”. NGS will markedly increase the clinician’sability to answer the first two questions, although the order in whichthese questions are answered will change. Patients are only rarelyinterested in the exact nature of a mutation they have, but are inter-ested in the consequences for their health (and for their relatives),such as the likelihood to develop a particular manifestations of a dis-order, how these manifestations evolve over time (natural history),and the need for specific health surveillance. Phenotypic researchwill be essential here: A detailed phenotype will allow researchersto compare and pool clinical findings in their patients and correlatethese findings with the results of NGS. The improvement affordedby NGS is that the molecular diagnosis will be more readily iden-tified and clinical evaluations can be performed in a directed way,avoiding stressful additional studies like biopsies or other invasiveprocedures.

Medical Genetics in NGS ResearchResearch techniques for the elucidation of the primary molecu-

lar etiology of Mendelian disease is at present more successful thanever, and NGS technology is the main factor that has acceleratedthis process. One may expect that in just a few years’ time, theprimary molecular basis for most currently recognized Mendeliandisorders will have been solved. In a research context, phenotyp-ing will remain important to identify mutations causing disorders.For disorders limited to a single-organ system, most phenotypingshould be performed by the relevant organ-specific specialists (e.g.,cardiologists for nonsyndromic congenital heart disease). However,for multisystem, pleiotropic disorders, the expertise of the medicalgeneticist will be essential.

A primary diagnostic label (e.g., “Marfan syndrome”) is not a suf-ficient level of clinical delineation to answer the patient’s questions.Patients want to understand the full range of phenotypic manifes-tations that they may manifest, and the clinician will want to knowwhere the patient is likely to fall on the spectrum of clinical severity.After all, the mild and severe end of the Marfan spectrum are verydifferent clinical scenarios. Most Mendelian disorders have signif-icant phenotypic heterogeneity with interindividual, intrafamilial,and interfamilial variability in expressivity and even penetrance.Patients (or parents) will ask whether they will have a mild or se-vere form of the disorder—and we cannot answer most of thesequestions, given the current state of knowledge.

Most implementations of NGS focus on Mendelian disorders.“Mendelian” indicates that a mutation (or two mutations for auto-somal recessive disorders) explains a major part of the phenotypein an individual. However, there is no protein that does not inter-act with other proteins, and interaction means that the structureand function of both proteins influence the resulting function andthus phenotype. In addition, most proteins undergo posttransla-tional processing and will not function in their primary, unprocessedform, or must be transported to a specific subcellular or extracellu-lar location. These processes are mediated by other proteins, whichare themselves subject to interindividual variation; they are termedmodifiers. Thus, it will be essential to identify modifier loci and cor-relate variation at those modifying loci to predict phenotype. Everyclinician has evaluated families in which a Mendelian disorder segre-gates, with substantial interindividual phenotypic variation. Thesedifferences in manifestations will not be clear from the early resultsof NGS.

Beyond Mendelian disorders and their modifiers are the oli-gogenic and subsequently polygenic or multifactorial disorders.Although it may be argued that all disorders are polygenic [Barabasi

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et al., 2011] (as the so-called “single-gene” disorders have modi-fiers), we use the term here to distinguish disorders for which thereare no variations at single loci that explain a large portion of thephenotypic variability of that disorder; yet, heritability is high, andepidemiology suggests that a substantial number of genes influencethe phenotype. The challenges here are largely unexplored; NGSmay be theoretically used to discover variants associated with thesephenotypes, but new analyses will be necessary. For rare disordersinherited in a dominant pattern, we currently ignore variants that arepresent in databases like the 1000 Genomes Project, as we can safelyassume that variants in these databases do not cause these disorders.However, these variants are the prime candidates for the molecularbasis of polygenic and multifactorial traits and their modifiers. Newbioinformatics and systems biology methods will be paramount insuch analyses. An important way to recognize these is by groupingpatients on the basis of their phenotype and selecting appropriatecontrols. The more carefully this phenotyping is performed, thehigher the chance a variant influencing the presence of this mani-festation will be found. New approaches to hypothesis-generatingstudies may also be needed. For example, one might analyze a largecohort of subjects with NGS sequence results to identify a subsetthat shares a particular set of variants. Then, those subjects can beclinically analyzed to see if they share phenotypic attributes. Espe-cially, for disorders in which phenotyping is extremely difficult andheterogeneity is large (e.g., psychiatric disorders) [Xu et al., 2011],such strategies may be extremely useful. This hypothesis-generatingmode of medical genetics research will necessitate a high level ofmultisystem clinical evaluation, something that can only be pro-vided by the medical geneticist.

The Future of Medical GeneticsBased on the above, it is clear that the future of medical genetics is

bright. For the foreseeable future, medical geneticists will be essen-tial in research to identify genes that are mutated in human diseaseusing NGS, dissect the role of modifiers, develop new approachesto NGS clinical research, and act as teachers for other specialties todisseminate these new diagnostic tools. As well, medical geneticistswill be necessary to develop databases and precise clinical languageto characterize phenotypes. Activities like the recent series of papersto define all terms describing the external human phenotype [Al-lanson et al., 2009] and other medical language ontology initiativesare essential to allow reliable comparisons of patients. As well, med-ical geneticists will need to adapt and broaden their skill sets. No

longer will it be sufficient to be only a phenotypic expert; the mostsuccessful medical geneticists will combine and complement theirclinical skills with expertise in molecular genetics (including NGS)and bioinformatics. Bioinformatic skills will be multifaceted—theywill be as much about phenotypic and medical informatics as theywill be about molecular data. Biomedicine is irrevocably movingtoward large-scale datasets, and the geneticist who can interrogateand analyze these data sets will have an enormous advantage overthose who do not have these skills.

ConclusionAs translational researchers operating at the boundaries of the

research laboratory and the clinic, we could not be more excitedand optimistic about the changes that are underway. We foresee anincrease in the need and demand for medical geneticists, especiallythose with additional molecular and bioinformatic skills. Our disci-pline is rapidly changing, and we believe that these changes will leadto dramatic improvements in our ability to diagnose and providetreatments for our patients. How we carry out these tasks will bequite different from how we currently approach both research andpatient care, and we are eager to jump in and lead the way in thecreation of these new approaches. NGS is the most powerful diag-nostic tool developed in medicine since the roentgenogram, and wepredict that its value and utility in clinical medicine will be enor-mous. We also conclude that the value of NGS can only be optimallyexploited if novel approaches to detailed phenotyping are integratedwith NGS results. Medical geneticists do not yet need to put phonenumbers of employment agencies in their mobiles. Instead, hospi-tals, academic medical centers, and research institutes will need toattract and support medical geneticists if they want to participate inthese exciting developments, or be left on the sidelines.

References

Allanson JE, Biesecker LG, Carey JC, Hennekam RC. 2009. Elements of morphology:introduction. Am J Med Genet 149A:2–5.

Barabasi AL, Gulbahce N, Loscalzo J. 2011. Network medicine: a network-based ap-proach to human disease. Nat Rev Genet 12:56–68.

Van Karnebeek CD, Scheper FY, Abeling NG, Alders M, Barth PG, Hoovers JM, KoevoetsC, Wanders RJ, Hennekam RC. 2005. Etiology of mental retardation in childrenreferred to a tertiary care center: a prospective study. Am J Ment Retard 110:253–267.

Xu B, Roos JL, Dexheimer P, Boone B, Plummer B, Levy S, Gogos JA, Karayiorgou M.2011. Exome sequencing supports a de novo mutational paradigm for schizophre-nia. Nat Genet 43:864–868.

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