Exome sequencing in an SCA14 family
demonstrates its utility in diagnosing
Anna Sailer, MD
Sonja W. Scholz, MD,
J. Raphael Gibbs, BSc
Arianna Tucci, MD
Janel O. Johnson, MSc
Vincent Plagnol, PhD
Jinhui Ding, PhD
Dena Hernandez, MSc
John Hardy, PhD
Howard J. Federoff,
Bryan J. Traynor, MD,
Andrew B. Singleton,
Henry Houlden, MD,
Objective: Genetic heterogeneity is common in many neurologic disorders. This is particularly true
for the hereditary ataxias where at least 36 disease genes or loci have been described for spino-
cerebellar ataxia and over 100 genes for neurologic disorders that present primarily with ataxia.
Traditional genetic testing of a large number of candidate genes delays diagnosis and is expen-
sive. In contrast, recently developed genomic techniques, such as exome sequencing that targets
only the coding portion of the genome, offer an alternative strategy to rapidly sequence all genes
in a comprehensive manner. Here we describe the use of exome sequencing to investigate a large,
5-generational British kindred with an autosomal dominant, progressive cerebellar ataxia in which
conventional genetic testing had not revealed a causal etiology.
Methods: Twenty family members were seen and examined; 2 affected individuals were clinically
investigated in detail without a genetic or acquired cause being identified. Exome sequencing was
performed in one patient where coverage was comprehensive across the known ataxia genes,
excluding the known repeat loci which should be examined using conventional analysis.
Results: A novel p.Arg26Gly change in the PRKCG gene, mutated in SCA14, was identified. This
variant was confirmed using Sanger sequencing and showed segregation with disease in the
Conclusions: This work demonstrates the utility of exome sequencing to rapidly screen heteroge-
neous genetic disorders such as the ataxias. Exome sequencing is more comprehensive, faster,
and significantly cheaper than conventional Sanger sequencing, and thus represents a superior
diagnostic screening tool in clinical practice. Neurology®2012;79:127–131
SCA ? spinocerebellar ataxia.
Genetic heterogeneity is common in inherited cerebellar ataxias with over 36 genetic loci
known to cause spinocerebellar ataxia (SCA) and over 100 genes that primarily present with
ataxia (http://neuromuscular.wustl.edu/ataxia/aindex.html). Only a relatively few hereditary
ataxia syndromes are associated with distinctive clinical features that indicate a particular ataxia
gene. Thus, even neurologists specialized in the care of patients with cerebellar disease are
frequently left with long lists of putative genes when dealing with such a patient. Screening
such a long list of genes is costly, as well as time-consuming, and genetic testing for some of the
rarer and difficult to sequence genes is not commercially available. The net result is that a
genetic diagnosis cannot be achieved in at least 40% of ataxia cases, even though a genetic cause
is heavily suspected.1
Recently developed genomic techniques provide the opportunity to screen large proportions
of the genome in a short time frame. Exome sequencing, which targets the coding portion of
From the Department of Molecular Neuroscience and Reta Lila Weston Laboratories (A.S., J.R.G., N.W.W., V.P., D.H., J.H., H.H.), Institute of
Neurology, University College London, London, UK; Department of Neurology (S.W.S., H.J.F.), Georgetown University, Washington, DC;
Laboratory of Neurogenetics (S.W.S., J.R.G., A.T., J.O.J., J.D., D.H., B.J.T., A.B.S.), National Institute on Aging, National Institutes of Health,
Bethesda, MD; Medical Research Council Prion Unit (H.H.), Department of Neurodegenerative Disease, University College London Institute of
Neurology, London, UK; and Department of Neurology (B.J.T.), Johns Hopkins Hospital, Baltimore, MD.
Study funding: Supported in part by the Intramural Research Program of the National Institute on Aging, NIH, Department of Health and Human
Services; project number: Z01 AG000958-08.
Go to Neurology.org for full disclosures. Disclosures deemed relevant by the authors, if any, are provided at the end of this article.
Editorial, page 112
Correspondence & reprint
requests to Dr. Houlden:
Copyright © 2012 by AAN Enterprises, Inc.
the genome, offers an affordable strategy to
comprehensively sequence all genes in the hu-
Here we report our genetic analysis of a
large British kindred with autosomal domi-
nant cerebellar ataxia. After excluding the
most common genes responsible for heritable
cerebellar ataxia by standard screening meth-
ods, we performed exome sequencing to find
the causative gene. In doing so, we demon-
strate that exome sequencing is an efficient
and cost-effective method for screening heter-
ogeneous diseases such as ataxia.
METHODS Samples. We identified a 5-generation family
with autosomal dominant SCA (19 affected, 15 unaffected indi-
viduals). Twenty family members were examined by a neurolo-
gist specialized in ataxias and 2 investigated for acquired causes
of ataxia (H.H.). In patient VI-14, the available diagnostic ataxia
gene screening was carried out for SCA1, SCA2, SCA3, SCA6,
SCA7, SCA8, SCA10, SCA11, SCA12, SCA15/16, SCA17,
POLG1, and DRPLA and a CGH array to reveal large structural
Standard protocol approvals, registrations, and patient
consents. All examined family members gave written informed
consent and the study was approved by the appropriate institu-
tional review board (IRB/ethics 06/N076).
Sequencing. Exome sequencing was performed in one affected
individual (IV-14) using NimbleGen Sequence Capture tech-
nology (2.1 Human Exome v1.0) according to the manufactur-
er’s instructions (Roche NimbleGen, Madison, WI). The
enriched libraries underwent 50 base pair, paired-end sequenc-
ing on a HiSeq2000 next-generation sequencing platform (Illu-
mina, San Diego, CA). The sequence data were aligned to the
reference human genome (UCSC hg18) and variant calling used
the Genome Analysis Toolkit (Broad Institute, Cambridge,
MA). After filtering PCR duplicates, previously reported variants
ncbi.nlm.nih.gov/snp, build 130) and synonymous variants were
removed. For potential disease-causing variants protein altera-
tion was predicted using SIFT (http://sift.jcvi.org/), presence ex-
cluded in 221 neurologically healthy control exomes, and the
variant was confirmed using Sanger sequencing (see appendix
e-1 on the Neurology®Web site at www.neurology.org for de-
tails). A total of 36 known ataxia genes were analyzed for se-
quencing quality in the exome data from 16 samples as described
RESULTS Clinical characteristics. The pedigree was
consistent with an autosomal dominant inheritance
pattern (figure e-1). Twenty family members were
examined, of which 10 showed signs of cerebellar
ataxia (table e-1). Acquired causes of ataxia were ex-
cluded by history and investigations in 2 individuals
(VI-10, VI-14). Mean age at symptom onset was 37
years (range 15–68 years), and the clinical presenta-
tion was a relatively pure cerebellar ataxia predomi-
nantly affecting lower limb coordination and
speech.17The age at symptom onset varied signifi-
cantly within the family; ataxia was only revealed on
neurologic examination in some patients. Symptom
progression was slow with an overall benign disease
course. Two affected members had unilateral mild
ptosis (IV-14, IV-10), and one patient had concomi-
tant mild parkinsonism (IV-7). Individual V-9 rap-
idly developed a very severe ataxia during pregnancy
at the age of 24 years (24 weeks gestational age); she
plateaued after delivery and has remained severely af-
fected since. She was considered phenotypically dif-
ferent from other family members, as detailed in the
Sequencing. We sequenced the exome of patient IV-
14, which provided ?10-fold coverage in 87% of her
exome. We identified 22,119 variants of which
1,061 were novel (844 homozygous, 217 heterozy-
gous). In this list, we noted a novel c.76A?G muta-
tion in exon 1 of the PRKCG gene, which is known
to cause SCA14.18The PRKCG gene had previously
been analyzed using DHPLC-based WAVE analysis
and found to be negative. This variant results in a
change from the polar basic amino acid arginine to a
hydrophobic, uncharged glycine residue at codon 26
(p.Arg26Gly). This variant is not reported in db-
SNP or the 1,000 genome project and is absent in
221 control exomes. Sanger sequencing confirmed
the mutation and demonstrated disease segregation
in all investigated family members (n ? 19; figure
e-1), giving a lod score of 5.7 for the mutation. Pa-
tient V-9 with a different ataxia phenotype did not
carry the mutation.
Ataxia locus screening. To assess the power of exome
sequencing in detecting mutations, we investigated
36 known recessive and dominant ataxia genes for
coverage and sequence alignment in the exome data
generated for 16 patients (table e-2). Of these 36
genes, 3 were not captured by the NimbleGen Se-
quence Capture Kit, and in an additional 5 genes the
disease-causing variants were noncoding and there-
fore not targeted in the capture array. Furthermore,
we found that the exome data had multiple align-
ment errors and poor coverage in the region of the
known CAG and other disease-causing repeats. The
length of expanded coding or noncoding repeats
therefore cannot be assessed accurately in the SCAs
using exome sequencing.
The other 21 ataxia genes showed good overall
coverage with an average read depth above 10 in all
of these genes and above 30 in 17 out of 21. Patho-
genic mutations in these genes are predominantly re-
ported to be coding nucleotide substitutions, small
indels, and splice site mutations, which can all be
reliably detected by exome sequencing.
Neurology 79July 10, 2012
DISCUSSION We used exome sequencing to iden-
tify the causative mutation in a large British family
with a pure autosomal dominant cerebellar ataxia,
the most heterogeneous group of inherited ataxia.
We had previously excluded 13 known ataxia genes
for which routine screening tests were available. Ex-
ome sequencing data analysis revealed a novel muta-
tion in the SCA14 gene (PRKCG) consistent with
this phenotype and the mutation showed complete
disease segregation among the 19 members of the
family screened. This novel c.76A?G (p.Arg26Gly)
mutation is predicted to be protein damaging and
occurs in a highly conserved nucleotide (figure 1C
and figure 2). We therefore conclude that the
p.Arg26Gly variant is disease-causing in this ataxia
Exome sequencing reveals a large number of vari-
ants (?20,000) normally present in any individual.
Trying to identify a disease-causing mutation in such
a large pool of variants is a major challenge. Filtering
out variants reported as nonpathogenic in databases
such as dbSNP is able to reduce the number of
potential mutations to several hundreds but also
harbors the risk of excluding pathogenic muta-
tions present in nonmanifesting individuals. To
further restrict the list of potential mutations link-
Figure 1 PRKCG gene and mutations
Shown is the PRKCG locus on chromosome 19. Previously reported nonsynonymous mutations are indicated using black font and the novel p.Arg26Gly
mutation is indicated in red.
Figure 2 The novel p.Arg26Gly PRKCG mutation
An electropherogram of the c.76A?G (p.Arg26Gly) mutation in patient IV-14 and sequence conservation plots at the
mutated site across different species are displayed.
Neurology 79July 10, 2012
age data from large informative kindreds is often
A potential application of exome sequencing is in
medical diagnostics for the screening of heteroge-
neous genetic diseases such as genetic ataxias. A large
number of known disease-causing genes can be inves-
tigated in a single experiment. Specifically looking in
such candidate genes greatly reduces the list of vari-
ants discovered by exome sequencing and will often
allow the accurate diagnosis in a single individual as
we have shown in our SCA14 patient.
Thus the method of exome sequencing offers a
potential alternative to currently available diagnostic
sequencing tests. Here we were able to show that the
majority of ataxia genes were well covered by this
technology. The ability to screen for mutations with
exome sequencing does vary depending mainly on
the mutational mechanism. Coding nucleotide sub-
stitutions, splice site changes, and small indels will be
covered19,20but by way of design, exome sequencing
does not routinely sequence noncoding regions and
is therefore not suitable for investigating intronic
variants unless in close proximity of a targeted exon.
However, custom-made libraries could be designed
to target these regions. Repetitive sequence stretches,
such as triplet repeats, cannot be determined accu-
rately, though advances in sequencing technologies
with longer read lengths may resolve this technical
issue in the future.
We demonstrated the utility of exome sequencing
to rapidly screen a large number of disease-causing
genes in a heterogeneous disease such as ataxia by
identifying a novel mutation in the PRKCG gene
known to cause SCA14. The majority of known
ataxia genes can be investigated with this method,
and therefore it harbors great potential as a tool for
rapid and comprehensive screening of such patients.
For a clinical diagnostic application, however, sensi-
tivity and specificity of exome sequencing would still
have to be validated in a larger ataxia cohort. Fur-
thermore, we anticipate that costs of exome sequenc-
ing will continue to decrease as next-generation
sequencing becomes more widely available (cur-
rently, ?$1,500 per patient for exome data produc-
tion and analysis) and thus the method will be more
cost-efficient than a series of individual gene tests,
each costing between hundreds and several thousand
dollars. As this happens, exome sequencing will be-
come an increasingly important and more widely
available test in the investigation of even the most
This study was designed and funding obtained by H.H., A.B.S., J.H.,
H.J.F., and D.H., H.H., A.S., N.W.W., and J.B. collected samples and
assessed patients clinically. A.S., S.W.S., and J.O.J. conducted experi-
ments. J.R.B., A.S., S.W.S., B.J.T., A.T., J.D., and V.P. performed data
analysis. The manuscript was written by H.H., A.S., S.W.S., and B.J.T.
The authors thank the patients and family who supported this work. They
also thank the following for grant support: The Medical Research Council
(H.H. and N.W.), The MSA Trust (A.S.), Ataxia UK (H.H.), and The
Wellcome Trust. This work was undertaken at UCLH/UCL, which re-
ceived a proportion of funding from the Department of Health’s NIHR
Biomedical Research Centres funding scheme.
A. Sailer, S. Scholz, J.R. Gibbs, A. Tucci, J. Johnson, N. Wood, V. Plag-
nol, H. Hummerich, J. Ding, J. Brown, D. Hernandez, J. Hardy, and H.
Federoff report no disclosures. B. Traynor received research support from
the ALS Association, The Packard Center for ALS Research, Microsoft
Research, Federazione Italiana Giuoco Calcio (FIGC), and The Myasthe-
nia Gravis Foundation. A. Singleton and H. Houlden report no disclo-
sures. Go to Neurology.org for full disclosures.
Received July 19, 2011. Accepted in final form December 14, 2011.
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