Rapid Genetic Diagnosis in Single-Gene Movement Disorders

Andrew B. Singleton, PhD*Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, USA

In this issue of Movement Disorders, Ruth Walkeret al. describe the genetic diagnosis of neuroacantho-cytosis disorders using a fairly new, but rapidlyprevailing, genetic method: exome sequencing.1 Thiswork highlights a standing problem in diseases with agreat deal of genetic heterogeneity: how to reach amolecular diagnosis. Perhaps more important, it illus-trates how this issue may be overcome quite routinelyin the near future.

Although not unique, the marked genetic heterogene-ity associated with many clinical presentations repre-sents a diagnostic problem that is pronounced inmovement disorders. Illustrative examples abound, asthere are at least 19 identified genetic causes of autoso-mal dominant ataxia,2 primary dystonia may be causedby known mutations at 10 different loci,3 recessiveparkinsonism by mutations in at least six differentgenes,4–9 and in the case of neuroacanthocytosis, thereare four genes known to contain disease-causing muta-tions, including a very large gene, VPS13A.1

Facing genetically heterogeneous, but clearly fami-lial, disorders in the clinic, a physician faces a decisionabout genetic testing that must balance cost, time,likelihood of a positive result, and the effect of anyresult on treatment, prognosis, and the patient’sfamily. A common approach is to test the most likelycandidate mutations or genes first, progressing steadilythrough alternates until a verdict is reached, or the mostplausible and testable candidate pool is exhausted. Thisiterative approach can be highly inefficient; it may takea substantial amount of time and money to find a muta-tion, and often, we still fail to identify the causal geneticlesion. There are many reasons for this: there remain alarge number of single-gene disorders for which we do

not know the genetic cause, many gene tests are notroutinely available, and variants are frequently discov-ered that are of unknown significance.

The quite broad phenotypic heterogeneity associatedwith a large number of gene mutations also presents ahurdle to identifying the specific genetic cause of clini-cal presentations. We know that single-gene movementdisorders can present with phenotypes outside of thenorm. Patients with a pathogenic LRRK2 mutationmost commonly present with typical PD10; however,some of the earliest LRRK2 families describedincluded patients with presentations such as amyotro-phy, dystonia, and dementia.11 Likewise, PLA2G6mutations, which were originally associated withinfantile neuroaxonal dystrophy, are known to alsocause dystonia parkinsonism.7,12 VCP mutations canlead to any one or a combination of frontotemporaldementia, inclusion body myopathy, amyotrophic lat-eral sclerosis, and Paget’s disease of bone.13 It is alsoreasonable to suspect that the same genetic lesion mayresult in different clinical presentations based ongenetic background, and this may be evident acrossdiverse populations. There already exists some supportof this in ataxia, where ATXN2 and ATXN3 expan-sion mutations, primarily described in Caucasianspinocerebellar ataxia patients, may present as a dopa-responsive and quite pure parkinsonism in patients ofAsian or African lineage.14–16

Given the challenges of substantial genetic and phe-notypic heterogeneity, what is needed is a way toscreen all gene candidates for a disorder, includingthose outside of the immediately plausible candidategene pool. Such a method needs to be quick and cost-effective and should ideally provide unequivocalresults that are easily interpretable. Exome sequencingpromises to best some of these challenges. In essence,this method aims to produce genetic sequence data onevery protein-coding DNA base pair of the humangenome, representing approximately 1/100th of thediploid genome, or approximately 70 million basepairs per individual. There are a number of approachesto this, but they each center on creating a library froma genomic DNA sample, which is enriched for thegenetic regions of interest, then analyzed using asecond-generation sequencer. This method generates

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*Correspondence to: Dr. Andrew B. Singleton, Laboratory ofNeurogenetics, National Institute on Aging, Bethesda, MD, USA;singleta@mail.nih.gov

Funding agencies: The author of this work is supported by theIntramural Research Program of the National Institute on Aging, NationalInstitutes of Health (part of the Department of Health and HumanServices) (project no.: Z01 AG000957-09).Relevant conflicts of interest/financial disclosures: Nothing to report.

Received: 25 November 2011; Accepted: 2 December 2011Published online in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/mds.24896

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billions of base pairs of information in quite shortorder. Sample preparation and sequencing takes �3weeks, but many samples can be sequenced in parallel.Following this, a computationally intensive, butincreasingly stable and accessible, analysis generates areport of all variants detected, their populationfrequencies, predicted effect on the protein product,and whether the variants have been described before.Current costs for an exome sequence run at approxi-mately $1,000. In the article by Ruth Walker et al.,this is exactly the approach that was taken. In thisstudy, the investigators performed exome sequencingin two unrelated individuals with neuroacanthocytosis.In both individuals, this method revealed compoundheterozygous mutations in VPS13A, a known geneticcause of this disorder.So, is exome sequencing the panacea to current limi-

tations faced in clinical testing? The information itprovides is certainly extensive, now quite accurate,and has been used to make genetic diagnosis, includ-ing unanticipated diagnosis.17 There are, however,some limitations to the method that bear considera-tion. In its current form, exome sequencing does notquite capture all of the protein-coding genome and isgenerally unable to capture or accurately sequenceapproximately 10% of this content. Exome sequencingis not particularly good at identifying repeat mutations(i.e., triplet repeats observed in spinocerebellar atax-ias, for instance), and although improving, methodsfor detecting gene deletions and duplications usingexome data are not well established.Currently, exome sequencing is not clinically vali-

dated and will likely require additional follow-up withestablished mutation discovery methods, once a candi-date variant has been identified. Despite these limita-tions, the comprehensive nature of exome means thatthis remains an attractive method for genetic testing;indeed, companies are now offering this as part of adiagnostic service.Given the breadth of sequencing afforded by this

method, it is also useful to consider the ramifications ofunanticipated genetic findings. What are the consequen-ces of a collateral discovery of a BRCA1 mutation in apatient without cancer or in identifying a carrier of arecessive mutation who is about to start a family? Oneof the earliest exome-sequencing studies provides anexample of this, finding not only the cause of Millersyndrome (DHODH mutation), a rare multiple malfor-mation disorder, but also showing genetically that oneof the patients had primary ciliary dyskinesia as a resultof DNAH5 mutations.18 These are clearly complexissues that will require a shift in our view of geneticdata (and perhaps genetic determinism); most impor-tant, this will require an informed discussion withpatients seeking genetic diagnosis using this method.One early consequence of diagnostic exome sequenc-

ing is the identification of a large number of variants

of unknown significance and an opportunity existshere. It is likely that clinical diagnostic services wouldbe the primary producer of sequences in monogenic orsuspected genetic disease, eventually outstripping thethroughput of research laboratories. What is called foris a centralized database of variants identified, linkedwith phenotypes, and this resource should be availablefor all of these clinical services to both data mine andcontribute to. This provides an opportunity to buildstatistical confidence in the pathogenic role (or not) ofidentified variants based on data accumulating world-wide. Though these are likely to be difficult waters tonavigate, particularly using clinical data for researchand maintaining subject anonymity, this seems aworthy aim, likely to identify new genes, new muta-tions, and perhaps new risk variants; ultimately, sucha service would greatly help genetic diagnostic accu-racy and risk prediction.Clearly, exome sequencing is already making an

impact in the research arena and is now the primarymethod in identifying novel genetic causes of disease.This has been discussed extensively elsewhere, so Iwill not go into details here, but there exists a bur-geoning body of gene-discovery literature that includesexamples in movement disorders (e.g., EIF4G1 andVPS35 mutations in PD and TGM6 mutations inSCA).19–22

Last, it is worth noting that exome sequencing likelyrepresents a half step toward a complete geneticsolution for genetic diagnosis, in the form of wholegenome sequencing. This method has already beenused to find new genetic causes of disease in theresearch setting.23 One would expect this approach torapidly find its way into the diagnostic field, probablyreplacing exome sequencing, because whole genomesequencing provides much more complete coverageand is better positioned to identify both structural andnonprotein coding mutations. Many of the challengesthat we will face with the implementation and inter-pretation of exome data will prepare us for wholegenome sequencing data, although we will likely haveto be more thoughtful about how we approach low-risk variability. The use of exome sequencing, and theinevitable transition to whole genome sequencing inclinical diagnosis, presents us with a host of opportu-nities, challenges, and food for thought.

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