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Cost-effectiveness of massively parallel sequencing for diagnosis of paediatric muscle diseases

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Childhood-onset muscle disorders are genetically heterogeneous. Diagnostic workup has traditionally included muscle biopsy, protein-based studies of muscle specimens, and candidate gene sequencing. High throughput or massively parallel sequencing is transforming the approach to diagnosis of rare diseases; however, evidence for cost-effectiveness is lacking. Patients presenting with suspected congenital muscular dystrophy or nemaline myopathy were ascertained over a 15-year period. Patients were investigated using traditional diagnostic approaches. Undiagnosed patients were investigated using either massively parallel sequencing of a panel of neuromuscular disease genes panel, or whole exome sequencing. Cost data were collected for all diagnostic investigations. The diagnostic yield and cost effectiveness of a molecular approach to diagnosis, prior to muscle biopsy, were compared with the traditional approach. Fifty-six patients were analysed. Compared with the traditional invasive muscle biopsy approach, both the neuromuscular disease panel and whole exome sequencing had significantly increased diagnostic yields (from 46 to 75% for the neuromuscular disease panel, and 79% for whole exome sequencing), and reduced the cost per diagnosis from USD$16,495 (95% CI: $12,413–$22,994) to USD$3706 (95% CI: $3086–$4453) for the neuromuscular disease panel and USD$5646 (95% CI: $4501–$7078) for whole exome sequencing. The neuromuscular disease panel was the most cost-effective, saving USD$17,075 (95% CI: $10,654–$30,064) per additional diagnosis, over the traditional diagnostic pathway. Whole exome sequencing saved USD$10,024 (95% CI: $5795–$17,135) per additional diagnosis. This study demonstrates the cost-effectiveness of investigation using massively parallel sequencing technologies in paediatric muscle disease. The findings emphasise the value of implementing these technologies in clinical practice, with particular application for diagnosis of Mendelian diseases, and provide evidence crucial for government subsidy and equitable access.
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PERSPECTIVE OPEN
Cost-effectiveness of massively parallel sequencing for
diagnosis of paediatric muscle diseases
Deborah Schoeld
1,2,3
, Khurshid Alam
2,4
, Lyndal Douglas
5,6
, Rupendra Shrestha
1
, Daniel G. MacArthur
7,8
, Mark Davis
9
,
Nigel G. Laing
9,10
, Nigel F. Clarke
6,11
, Joshua Burns
6,11,12
, Sandra T. Cooper
6,11
, Kathryn N. North
2,4,6
, Sarah A. Sandaradura
5,6,11
and
Gina L. OGrady
6,11,13
Childhood-onset muscle disorders are genetically heterogeneous. Diagnostic workup has traditionally included muscle biopsy,
protein-based studies of muscle specimens, and candidate gene sequencing. High throughput or massively parallel sequencing is
transforming the approach to diagnosis of rare diseases; however, evidence for cost-effectiveness is lacking. Patients presenting
with suspected congenital muscular dystrophy or nemaline myopathy were ascertained over a 15-year period. Patients were
investigated using traditional diagnostic approaches. Undiagnosed patients were investigated using either massively parallel
sequencing of a panel of neuromuscular disease genes panel, or whole exome sequencing. Cost data were collected for all
diagnostic investigations. The diagnostic yield and cost effectiveness of a molecular approach to diagnosis, prior to muscle biopsy,
were compared with the traditional approach. Fifty-six patients were analysed. Compared with the traditional invasive muscle
biopsy approach, both the neuromuscular disease panel and whole exome sequencing had signicantly increased diagnostic yields
(from 46 to 75% for the neuromuscular disease panel, and 79% for whole exome sequencing), and reduced the cost per diagnosis
from USD$16,495 (95% CI: $12,413$22,994) to USD$3706 (95% CI: $3086$4453) for the neuromuscular disease panel and USD
$5646 (95% CI: $4501$7078) for whole exome sequencing. The neuromuscular disease panel was the most cost-effective, saving
USD$17,075 (95% CI: $10,654$30,064) per additional diagnosis, over the traditional diagnostic pathway. Whole exome sequencing
saved USD$10,024 (95% CI: $5795$17,135) per additional diagnosis. This study demonstrates the cost-effectiveness of
investigation using massively parallel sequencing technologies in paediatric muscle disease. The ndings emphasise the value of
implementing these technologies in clinical practice, with particular application for diagnosis of Mendelian diseases, and provide
evidence crucial for government subsidy and equitable access.
npj Genomic Medicine (2017) 2:4 ; doi:10.1038/s41525-017-0006-7
INTRODUCTION
Childhood-onset muscle disorders present with hypotonia and
delay of gross motor milestones. Congenital muscular dystrophies
(CMD) are characterised by dystrophic features on skeletal muscle
biopsy and commonly by elevated creatine kinase (CK) levels in
blood. The (non-dystrophic) congenital myopathies, of which
nemaline myopathy (NM) is a subtype, are characterised by
specic morphological features on muscle biopsy. Massively
parallel sequencing technology is transforming the diagnostic
evaluation of this heterogeneous group of disorders.
Increasing evidence supports improved diagnostic success
using both neuromuscular gene panels and whole exome
sequencing (WES), with diagnosis rates ranging from 49 to
83%.
14
However, there is limited evidence regarding the relative
cost-effectiveness of diagnostic evaluation using massively parallel
sequencing technology, compared with traditional diagnostic
work-up using muscle biopsy, histological and biochemical
analyses of muscle specimens, followed by Sanger sequencing
of candidate genes. With genomic technologies rapidly entering
clinical practice, evidence for cost effectiveness is crucial in many
countries to obtain government subsidy for this new technology
and thus ensure equity of access.
We have retrospectively evaluated the diagnostic outcomes for
a cohort of 56 patients with childhood-onset muscle diseases
(Supplementary Table e1) comparing traditional diagnostic
techniques with massively parallel sequencing technologies, to
evaluate the economic impact of a shift toward molecular-based
diagnostics.
RESULTS
Patient cohort and diagnostic outcomes
We identied a cohort of 58 patients (40 CMD patients and 18 NM
patients), ascertained over 15 years from a publically funded,
Received: 7 October 2016 Revised: 3 January 2017 Accepted: 17 January 2017
1
Faculty of Pharmacy, University of Sydney, Sydney, NSW, Australia;
2
Murdoch Childrens Research Institute, Melbourne, VIC, Australia;
3
Garvan Institute for Medical Research,
Darlinghurst, NSW, Australia;
4
Faculty of Medicine, Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia;
5
Department of Clinical Genetics, Childrens
Hospital at Westmead, Locked Bag 4001, Sydney, NSW, Australia;
6
Institute for Neuroscience and Muscle Research, Kids Research Institute, Childrens Hospital at Westmead,
Sydney, NSW, Australia;
7
Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA;
8
Program in Medical and Population Genetics, Broad Institute
of Harvard and MIT, Cambridge, MA, USA;
9
Department of Diagnostic Genomics, PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, WA, Australia;
10
Centre for
Medical Research University of Western Australia, Harry Perkins Institute of Medical Research, Nedlands, WA, Australia;
11
Faculty of Medicine, Discipline of Paediatrics and Child
Health, University of Sydney, Sydney, NSW, Australia;
12
Sydney Childrens Hospitals Network (Randwick and Westmead), University of Sydney, Sydney, NSW, Australia and
13
Paediatric Neuroservices, Starship Childrens Health, Auckland, New Zealand
Correspondence: Sarah A. Sandaradura (sarah.sandaradura@health.nsw.gov.au)
Sarah A. Sandaradura and Gina L. OGrady contributed equally to this work
www.nature.com/npjgenmed
Published in partnership with the Center of Excellence in Genomic Medicine Research
tertiary pediatric Australian neuromuscular centre. Demographic
characteristics of the cohort are characterized in Table 1, and
genetic diagnoses in Supplementary Table e1. Fifty-two patients
were the index case and six were affected siblings. The mean
duration of the diagnostic odyssey was 7.7 years (range 2 months
to 26 years). Two CMD patients did not consent to further genetic
evaluation and were excluded from further analysis. Thus, 56
patients were included in the nal cohort.
Using traditional diagnostic approaches (Fig. 1), a genetic
diagnosis was achieved for 46% of patients (26/56), including two
patients diagnosed by chromosomal microarray. The 30 patients
who remained undiagnosed after traditional diagnostic investiga-
tions were investigated using WES, with the exception of three
patients who had been investigated by the neuromuscular disease
(NMD) panel without WES, and one sibling who had neither WES
nor panel, while WES was performed on the proband (Fig. 2). This
resulted in 18 additional diagnoses, including four patients with
variants in newly identied disease genes: two CMD siblings
contributed to identication of PIGY
5
and two siblings with NM
contributed to identication of LMOD3.
6
Two patients had variants
in recently published genes (MICU1 and GFPT1).
Cost-effectiveness from economic analysis
A comparative analysis of the combined CMD and NM cohort was
performed, representative of a patient cohort for whom there was
early access to WES or panel testing, prior to muscle biopsy. While
both the NMD panel and WES were superior (i.e. less costly with a
higher number of diagnoses) compared to the traditional diagnostic
pathway, the NMD panel was the most cost effective, with an
incremental cost saving per diagnosis of AUD$23,390 (95% CI:
$14,595$41,184) compared to traditional diagnostic investigations.
Although less cost-effective than the NMD panel, WES offered a cost
saving per diagnosis of AUD$13,732 (95% CI: $7938$23,473)
(Supplementary Table e2). Comparative costs per diagnosis and
cost-effectiveness planes are presented in Figs. 3and 4.
The average cost of traditional diagnostic investigation was
AUD$10,491 (95% CI: $9115$11,848) per patient, and AUD
$22,596 (95% CI: $17,004$31,498) per successful diagnosis.
The NMD panel provided 16 more diagnoses than traditional
investigations (42/56 patients, 75%), a 28.6% (95% CI: 17.841.1%)
increase in diagnostic rates and cost $6683 (95% CI: $5276$7947)
less per patient. The increase in diagnostic rates and the reduction
in the cost per patient were statistically signicant at 5% level of
signicance. The NMD panel cost less than a quarter of the cost of
the traditional pathway per diagnosis AUD$5077 (95% CI: $4228
$6100). WES achieved two more diagnoses than the NMD panel at
a diagnostic rate of 79% (44/56 patients), and a 32.1% (95% CI:
21.444.5%) increase compared to the traditional pathway and
cost signicantly less per patient, while costing about one-third of
the cost of the traditional pathway per diagnosis AUD$7734 (95%
CI: $6166$9696) (Supplementary Table e2, Fig. 3).
The ndings were similar when the CMD and NM cohorts were
analysed separately (Supplementary Table e2). The cost-
effectiveness plane for the CMD cohort is shown in Fig. 4b and
for the nemaline cohort in Fig. 4c.
The costs were re-calculated for probands only. As expected,
there was a slightly increased cost of diagnosis (Supplementary
Table e3), reecting the reduced cost of investigating an affected
sibling with Sanger sequencing when the pathogenic variant
had already been identied in the family. However, given the small
number of siblings in this cohort (n= 6), there was not a statistically
signicant difference when compared with the total cohort.
DISCUSSION
This study demonstrates that using either a NMD panel or WES,
prior to biopsy, is less costly and more effective than traditional
diagnostic investigation. The diagnostic efcacy increased from
46% for traditional investigation, to 75% for the NMD panel and
79% for WES, and the total average diagnostic expenditure per
patient was reduced from AUD$10,491 to AUD$3808 for the NMD
panel and AUD$6077 for WES. The total average cost per diagnosis
was reduced from AUD$22,596 to AUD$5077 for the NMD panel
and AUD$7734 for WES.
Patients undiagnosed by traditional means were investigated
with WES. The additional diagnoses in this cohort highlight some
key advantages of massively parallel sequencing, namely,
identication of unexpected diagnoses or a typical phenotypes,
for example, the patient diagnosed with a congenital myasthenic
syndrome; and variants in large genes such as TTN,NEB, and RYR1,
which have been technically difcult and prohibitively expensive
to Sanger sequence. Overall, fewer investigations were performed
in the nemaline cohort as part of the traditional diagnostic work
up, as the diagnosis of NM was made on muscle ultrastructural
analysis and the commercial cost of NEB sequencing (AUD
$7952.00) was not nancially viable. Identication of NEB as the
causative gene in almost half of the nemaline patients diagnosed
via targeted massively parallel sequencing panel (6 of 11 patients)
highlights the power of this approach for these patients.
Although WES was more expensive than NMD panel, it permits
retrospective data analysis as new genes are identied and offers
the potential to identify novel genetic causes of disease,
particularly when coupled with a research programme. Within
this cohort, two novel causes of disease (PIGY and LMOD3) were
identied.
5,6
This study was, however, intended to reect clinical
practice, rather than a research setting, and commercial costs have
therefore been selected for WES and NMD panel. We stress the
importance of WES as a gene discovery research tool that
underpins expansion of NMD gene panels, but note that our
calculations do not include the considerable research costs
involved in performing the functional studies necessary to verify
pathogenicity of novel disease genes or novel genetic mechan-
isms (for example, splicing aberrations). Important for centres
without a parallel research programme, the NMD gene panel
offered an excellent diagnostic yield (16 further diagnoses) and
improved cost-effectiveness when compared with the traditional
diagnostic pathway (an incremental cost saving of AUD$23,390
per additional diagnosis).
Our molecular approach to diagnosis prescribes a list of limited
investigations recommended prior to investigation with WES or
NMD panel (Table 2), focusing on the exclusion of diagnoses not
identied by these technologies. These include chromosomal
microarray to exclude large-scale copy number variation, or
tailored genetic testing for trinucleotide repeat disorders, not
Table 1. Demographic characteristics of the cohort
Characteristics Number (%)
Sex
Male 30 (53.6%)
Female 26 (46.4%)
Clinical diagnosis
Congenital muscular dystrophies (CMD) 38 (67.9%)
Nemaline myopathy (NM) 18 (32.1%)
Parental consanguinity 7 (12.5%)
Age at onset
Birth 34 (60.7%)
1
st
Year 11 (19.6%)
2
nd
Year 8 (14.3%)
>2
nd
Year 3 (5.4%)
Cost-effectiveness of massively parallel sequencing
D Schoeld et al
2
npj Genomic Medicine (2017) 4 Published in partnership with the Center of Excellence in Genomic Medicine Research
easily detected using massively parallel sequencing. In addition to
cost savings, a key benet of the molecular approach to diagnosis
is avoidance of procedures in some patients. Muscle biopsy, and
the associated general anaesthetic, is a potential risk in infants and
children with severe weakness and impaired respiratory function,
and has a risk of malignant hyperthermia reaction in some
patients. However, muscle biopsy will still be required as a second-
tier investigation in patients with variants in novel or recently
described disease genes, and has signicant utility for diagnosis of
nemaline patients with variants of uncertain signicance in NEB.
Morphological conrmation of nemaline bodies on biopsy and our
research-based utilisation of RNA-seq from muscle biopsy speci-
mens have been essential in establishing pathogenicity of splice
variants, particularly in NEB (Cummings et al., personal
communication).
In summary, our diagnostic evaluation and economic analysis
provide support for molecular diagnosis using massively parallel
sequencing technologies as a rst-tier diagnostic investigation for
paediatric NMD, replacing traditional investigation with muscle
biopsy and candidate gene sequencing. WES is a valuable tool in
the investigation of undiagnosed cases, particularly when coupled
with a research-based gene discovery programme. A molecular
approach to diagnosis also has the potential to decrease the
duration of the diagnostic odyssey. Of note, ve families in this
study had multiple affected siblings. Early genetic diagnosis has
signicant implications for families and society, including
informed reproductive choices, a potential impact on genetic
counselling and fertility services, and the possibility that some
families may chose not to have a second affected child.
Quantication of these downstream impacts was outside the
scope of this study, but warrants assessment in a future
prospective study. This study offers evidence to support the
cost-effectiveness of massively parallel sequencing techniques in
the diagnosis of highly heterogeneous paediatric muscle disorders
and to advocate for equitable access to genetic diagnosis in
clinical practice.
METHODS
This study was a retrospective cohort analysis conducted in a
publically funded Australian paediatric hospital. Patients referred
with suspected CMD or NM over a 15-year period (19982013)
were identied through the clinical records of the Neuromuscular
Clinic at the Childrens Hospital at Westmead, and the Institute for
Neuroscience and Muscle Research (Sydney, Australia) Biospeci-
men Bank. Patients referred to the clinic for a second opinion, and
patients whose diagnostic evaluation had been performed else-
where were excluded to ensure complete ascertainment of costs
(n= 3).
CMD patients were included in the study if their clinical
presentation was consistent with CMD, CK levels were elevated
(>200 IU/L) and the muscle biopsy showed dystrophic changes or
showed non-specic myopathic ndings, provided the clinical
criteria were met.
2,7
NM patients were included if their clinical
Fig. 1 Proposed diagnostic algorithms. ashows the traditional diagnostic algorithm based on muscle biopsy and protein-based studies of
muscle biopsy specimens, followed by candidate gene sequencing. Complementary investigations were selected in the cohort by treating
clinicians based on the presenting phenotype. bshows the proposed molecular approach to diagnosis. Complementary investigations were
included dependent on the presenting phenotype, based on Table 2. Molecular investigation using either a NMD gene panel or WES was
performed prior to muscle biopsy. NMD neuromuscular disease, WES whole exome sequencing, WGS whole genome sequencing, SMA spinal
muscular atrophy, FSHD facioscapular humeral dystrophy, DMD Duchenne muscular dystrophy
Cost-effectiveness of massively parallel sequencing
D Schoeld et al
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Published in partnership with the Center of Excellence in Genomic Medicine Research npj Genomic Medicine (2017) 4
presentation was consistent with a congenital-onset or childhood-
onset myopathy and muscle biopsy showed nemaline rods on
gomori trichrome stain and/or electron microscopy.
8
Patients had been investigated using a traditional diagnostic
approach
2,7,8
(Fig. 1), which routinely included measurement of
CK, muscle biopsy, and histological analysis. Further investigations
were performed at the discretion of the treating clinician
according to the patients phenotype. Patients who remained
undiagnosed despite candidate gene sequencing were offered
WES or a massively parallel sequencing-based neuromuscular
gene panel (NMD panel). WES was performed by the Broad
Institute, on an Illumina HiSeq2000 and/or 2500s, using a
previously published protocol.
9
The NMD panel represents a
commercially available 464 neuromuscular gene panel offered by
PathWest Laboratory, Australia (Supplementary Table e4). The
duration of the diagnostic trajectory was calculated as the time
from onset of symptoms until a genetic diagnosis was reported, or
an uninformative WES report was issued.
Approval for all aspects of this study was obtained from the
Human Research Ethics Committee of the Sydney Childrens
Hospitals Network (Approval No: 10.CHW.45). Written informed
consent was obtained from all participants for further genetic
testing.
Economic analysis
Economic analysis was performed from the payersperspective
and covered the period from the referral of patients with
suspected CMD or NM to a successful genetic diagnosis with
one of the pathways (the traditional diagnostic pathway, the NMD
panel, or WES) or the return of the WES results if no genetic
diagnosis was achieved. Cost data reect the 2016 commercial
charges to the hospital for all diagnostic investigations and
procedures, extracted from the medical records of each patient.
The current cost was identied either from the Australian Medicare
Benets Schedule, or from the hospital, research, or commercial
laboratory which provided the test (Supplementary Table e5).
These costs include sequencing plus bioinformatics and analysis.
The cost of regular clinic assessment was not included, as patients
were seen regularly in clinic regardless of whether or not they had
a genetic diagnosis. Geneticists and genetic counsellors work
routinely in our clinic and were available to all patients. Genetic
counselling for families undergoing WES potentially requires
additional resources, but could not be quantied in this analysis.
Costs in overseas currencies were converted into Australian dollars
(AUD$) (AUD$1 = USD$0.73 = GBP£0.50; Jan 2016).
We compared the traditional diagnostic pathway with two
alternative diagnostic pathways (counterfactual pathways), either
NMD gene panel or WES. The counterfactual pathways assumed
molecular investigation with NMD gene panel or WES prior to
muscle biopsy (Fig. 1). Table 2details a list of investigations
recommended prior to the NMD panel or WES diagnostic
approach. Supplementary Tables e2and e4detail the costs of
investigations.
Fig. 2 Case ascertainment and investigation. Ascertainment and
diagnostic outcomes for the combined CMD and NM cohorts. a
Three patients were investigated using only the NMD panel, four
patients had both NMD panel and WES, and one sibling had neither
NMD panel or WES while investigations were performed on the
proband. bDiagnostic outcomes for the NMD panel are derived
from data obtained from WES and based on previous studies
comparing the diagnostic efcacy of WES and NMD panel testing,
given only a small subset (n=4) were investigated using both WES
and the NMD panel. An assumption is made that patients diagnosed
using candidate gene sequencing would also be diagnosed using
CMA plus NMD panel or WES. CMD congenital muscular dystrophy,
CMA chromosomal microarray, NM nemaline myopathy, NGS next-
generation sequencing, NMD neuromuscular disease, WES whole
exome sequencing
Fig. 3 Cost savings by investigation type. Cost per diagnosis ($AUD)
of the traditional diagnostic pathway compared with NMD gene
panel and WES. Costs are divided by laboratory investigations,
genetic testing, procedural costs, and muscle biopsy and medical
imaging, demonstrating cost savings using a molecular approach to
diagnosis, predominantly in genetic testing costs and procedural
costs
Cost-effectiveness of massively parallel sequencing
D Schoeld et al
4
npj Genomic Medicine (2017) 4 Published in partnership with the Center of Excellence in Genomic Medicine Research
1) Traditional diagnostic pathway: Costing includes all diagnostic
investigations, procedures, and assessments for the traditional
diagnostic assessment, followed by Sanger sequencing of candidate
genes in genomic DNA or mRNA/cDNA extracted from biopsy
specimens, and conrmation of segregation of dominant and
recessive variant(s) in gDNA extracted from the parents.
2) NMD gene panel: Costing includes recommended investigations
(Table 2), if these investigations had been performed as part of the
traditional diagnostic pathway for the proband, DNA extraction, and
national shipping, a commercially available 464 gene neuromuscular
panel (PathWest Laboratory, Australia, AUD$1100.00) (Supplementary
Table e4), Sanger sequencing of identied causal variant(s) in the
proband, and conrmation of segregation of dominant and recessive
variant(s) in the parents.
3) WES: Costing includes recommended investigations (Table 2) if
performed on the proband as per the traditional diagnostic pathway,
DNA extraction and international shipment, Sanger conrmation of
the identied causal variant(s) in the proband, and conrmation of
segregation of dominant and recessive variant(s) in the parents. The
cost of singleton WES was included if the resulting genetic diagnosis
was a likely pathogenic variant in a known NMD gene, which would
be detected by the NMD panel. The cost of trioWES was included
for patients who remained undiagnosed and for patients with
diagnoses made by WES if the pathogenic gene was not included
in the NMD panel (i.e., Family 4 with variants in PIGY). The cost for WES
was based on an average commercially available price of AUD
$2600.00 for a singleton or AUD$7100 for a trio. Of note, the cost of a
research programme necessary to take the novel genetic ndings to
publication is not included in this calculation.
An increased cost of Sanger sequencing was used for patients
with NM due to variants in nebulin (NEB),reecting the likely need
to check segregation of a minimum of three candidate variants in
these patients, as NEB is highly polymorphic and individuals
commonly have multiple Class 3 variants. No value for WES or
NMD panel was included for siblings who could be diagnosed by
Sanger sequencing of the known familial variant(s), or for the
two patients with microdeletions detectable by chromosomal
microarray.
We calculated the average cost per patient, average cost per
genetic diagnosis, and incremental cost per additional diagnosis
that was achieved by the NMD panel and WES diagnostic
pathways compared with the traditional diagnostic pathway. To
estimate the uncertainty associated with outcomes, we created
1000 replicated data sets using bootstrap simulations and
estimated 95% condence intervals (CIs) for the outcomes using
the percentile method.
10
Results are presented as scatterplots on a
cost-effectiveness plane. All analyses were performed in Microsoft
Excel except for bootstrap simulations and 95% CIs which were
calculated in SAS version 9.4.
In calculating the diagnostic rates for WES and NMD panel
diagnostic pathways, for economic evaluation, two assumptions
Fig. 4 Cost-effectiveness of NMD panel or WES compared with the traditional diagnostic pathway. aCost saving per additional diagnosis, for
the combined CMD and NM cohort, for WES and NMD panel compared with the traditional diagnostic pathway. bCost saving per additional
diagnosis for the CMD cohort. cCost saving per additional diagnosis for the NM cohort
Cost-effectiveness of massively parallel sequencing
D Schoeld et al
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Published in partnership with the Center of Excellence in Genomic Medicine Research npj Genomic Medicine (2017) 4
needed to be made. It was not nancially viable to re-investigate
all cases previously diagnosed by candidate gene sequencing
using WES or the NMD panel. It was thus assumed that all
diagnoses in known CMD or myopathy genes previously identied
using candidate gene sequencing would be detected by WES or
the NMD panel. It was not viable to investigate undiagnosed
patients with both WES and NMD panel, and it was therefore
assumed that all patients diagnosed by WES would have been
diagnosed using the NMD panel if the gene is included in the
panel; and patients diagnosed by NMD panel would be diagnosed
by WES. These assumptions are based on our previous studies
which have demonstrated comparable efcacy of WES and the
NMD panel for identifying variants in known NMD genes.
2,9
ACKNOWLEDGEMENTS
This study was supported by the National Health and Medical Research Council of
Australia (1022707, 1031893, N.F.C,N.G.L., K.N.N, J.B.; APP1002147, N.G.L.;
GNT1113531, K.N., N.G.L., S.T.C., D.S.; 1056285, G.L.O; 1075451, S.A.S; 1048816 S.T.C;
1080587 N.G.L., S.T.C., D.G.M., N.F.C., K.N.N.), NHMRC-European Union Collaborative
Research Grant Scheme (1055131, K.N., J.B) and the Victorian Governments
Operational Infrastructure Support Program. G.L.O. received funding from Muscular
Dystrophy NSW and the Royal Australasian College of Physicians. Exome sequencing
was supported by grants from the NIH National Human Genome Research Institute
(Medical Sequencing Program grant U54 HG003067 to the Broad Institute principal
investigator).
AUTHOR CONTRIBUTIONS
D.S.: Study design and concept, data acquisition and analysis (health economic
analysis), drafting and revision of manuscript for content, obtaining funding (NHMRC
GNT1113531). K.A.: Data analysis (health economic analysis), drafting and revision of
manuscript for content. L.D.: Data acquisition (costing data) and manuscript revision.
R.S.: Data analysis (health economic analysis), drafting and revision of manuscript for
content. D.G.M.: Data acquisition, analysis and intepretation (co-director of the Broad
Institute Center for Mendelian Genomics facilitating whole exome sequencing),
revision manuscript for content, obtaining funding (NHMRC 1080587; NIH National
Human Genome Research Institute, Medical Sequencing Program grant U54
HG003067). M.D.: Data analysis and interpretation (NMD panel), revision of
manuscript for content. N.G.L.: Study design and concept, data analysis and
interpretation (NMD panel), obtaining funding (NHMRC 1022707, 1031893,
GNT1113531, 1080587), revising manuscript for content. N.F.C.: Study design and
concept, data analysis and interpretation, obtaining funding (NHMRC 1022707,
1031893, 1080587), study supervision. N.F.C. is sadly deceased and has not signed
this contribution form. J.B.: Study design and concept, revising manuscript for
content, obtaining funding (NHMRC 1022707, 1031893; NHMRC-European Union
Collaborate Research Grant Scheme 1055131). S.T.C.: Data analysis and interpretation,
drafting/revising manuscript for content, study design and concept, obtaining
funding (NHMRC GNT1113531, 1048816, 1080587), study supervision. K.N.N.: Study
design and concept, data analysis and interpretation, revising manuscript for content,
obtaining funding (NHMRC 1022707, 1031893, GNT1113531, 1080587; NHMRC-
European Union Collaborate Research Grant Scheme 1055131 and the Victorian
Governments Operation Infrastructure Support Program), study supervision. S.A.S.:
Analysis of whole exome sequencing data, reveiw of patients, collation of cost data,
lead role in the drafting of the manuscript and preparation of tables. S.S. received a
stipend from the NHMRC (1075451). G.L.O.: Analysis of whole exome sequencing
data, review of patients, collation of cost data, lead role in the drafting of the
manuscript and preparation of gures and tables. G.L.O. received a stipend from the
NHMRC (1056285), Muscular Dystrophy NSW, and the Royal Australasian College of
Physicians.
COMPETING INTERESTS
The funding bodies provided nancial support for materials and salary, but had no
role in design and conduct of the study, collection, management, analysis or
interpretation of the data, or in preparation or review of the manuscript. S.S. and G.O.
had full access to all of the data in the study and take responsibility for the integrity
of the data and the accuracy of the data analysis. The authors have no competing
interests to declare.
Table 2. Essential and recommended investigations used for assignment of counterfactual costs
Neonatal/early infantile presentation Early childhood presentation
Essential investigations Essential investigations
Blood collection × 2 Blood collection × 2
Creatine kinase Creatine kinase
Lactate Lactate
Ammonia Ammonia
Urine metabolic screen Urine metabolic screen
DNA extraction and storage DNA extraction and storage
Chromosomal microarray Chromosomal microarray
Investigations which might still be considered depending on presentation Investigations which might still be considered depending on presentation
Very long chain fatty acids Thyroid function studies
Thyroid function studies Myotonic dystrophy (DM1)
Plasma amino acids SMN1 exon 7 copy number (SMA)
Myotonic dystrophy MRI brain +/anaesthetic and day stay admission
SMN1 exon 7 copy number (SMA) Muscle MRI scan +/anaesthetic and day stay admission
Prader Willi syndrome Dystrophin MLPA
Acetylcholine receptor antibodies Ophthalmology review
Antibody screen for congenital infection Connective tissue dysplasia clinic review
Lumbar puncture with lactate, amino acids, and neurotransmitters
Head ultrasound scan
MRI brain +/anaesthetic and day stay admission
Ophthalmology review
MLPA multiplex ligation-dependent probe amplication, SMA spinal muscular atrophy
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npj Genomic Medicine (2017) 4 Published in partnership with the Center of Excellence in Genomic Medicine Research
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Supplementary Information accompanies the paper on the npj Genomic Medicine website (doi:10.1038/s41525-017-0006-7).
Cost-effectiveness of massively parallel sequencing
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Published in partnership with the Center of Excellence in Genomic Medicine Research npj Genomic Medicine (2017) 4

Supplementary resource (1)

... The use of genetic testing as a first-line test shortens the diagnostic odyssey and minimizes diagnostic delays. Patients can also avoid unnecessary and/or invasive investigations, such as muscle or nerve biopsies (9)(10)(11)(12). When Haskell et al. reviewed undiagnosed neuromuscular patients who had undergone investigations prior to targeted exome sequencing, they found that nearly 40% of the procedures, including muscle biopsy, were not helpful with pinpointing a molecular diagnosis in the patients (13). ...
... In such individuals, the diagnostic yield of gene panels is good, therefore, making gene panels suitable as the first-tier approach (13, 28). This gene panel-first strategy has recently been shown to be costeffective (10,64). If the gene panels are not informative, the second tier test is WES/WGS (65). ...
... For muscle disorders, diagnostic yield can range from 13 to 79% depending on the selected study cohort and gene panel testing techniques (10,12,13,30,84,87,89). For well-defined myopathies, a narrower gene panel may have comparable diagnostic yields as a broader gene panel (13). ...
Article
Full-text available
The diagnosis of inherited neuromuscular disorders is challenging due to their genetic and phenotypic variability. Traditionally, neurophysiology and histopathology were primarily used in the initial diagnostic approach to these conditions. Sanger sequencing for molecular diagnosis was less frequently utilized as its application was a time-consuming and cost-intensive process. The advent and accessibility of next-generation sequencing (NGS) has revolutionized the evaluation process of genetically heterogenous neuromuscular disorders. Current NGS diagnostic testing approaches include gene panels, whole exome sequencing (WES), and whole genome sequencing (WGS). Gene panels are often the most widely used, being more accessible due to availability and affordability. In this mini-review, we describe the benefits and risks of clinical genetic testing. We also discuss the utility, benefits, challenges, and limitations of using gene panels in the evaluation of neuromuscular disorders.
... Previous studies have not used a similar control group of patients (16). Many of the previous studies were modeled with diagnostic scenarios in the same study cohort (17)(18)(19)(20)(21) or using a hypothetical WES trajectory (22). In addition, only a few studies were conducted in Europe (10,22). ...
... In addition, only a few studies were conducted in Europe (10,22). Also, previous studies mainly investigated cost-effectiveness of WES in pediatric J o u r n a l P r e -p r o o f 10 patients with any suspected monogenic disorders (6,(19)(20)(21) or with specific disorders, such as epilepsy (17) or muscle disorders (18). The finding that clinical visits and genetic tests were the main drivers of costs in both study groups are in line with previous studies, in pediatric cohorts (10) and mixed cohorts of children and adults (23) with complex neurological problems. ...
... The finding that clinical visits and genetic tests were the main drivers of costs in both study groups are in line with previous studies, in pediatric cohorts (10) and mixed cohorts of children and adults (23) with complex neurological problems. Most of the previous studies have reached incremental cost savings per additional diagnosis when WES was used as a firstline test (18,19,21). In a population-based study by Howell et al. (17) WES also yielded cost savings per additional diagnosis only when WES was targeted early and metabolic testing was limited compared to standard care without WES in patients with severe infantile epilepsies. ...
Article
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Objectives To clarify the diagnostic utility and the cost-effectiveness of whole-exome sequencing (WES) as a routine early-diagnostic tool in children with progressive neurological disorders. Methods Patients with infantile-onset severe neurological diseases or childhood-onset progressive neurological disorders were prospectively recruited to this WES study, in the pediatric neurology clinic at Helsinki University Hospital during 2016–2018. A total of 48 patients underwent a singleton WES. A control group of 49 children underwent traditional diagnostic examinations and were retrospectively collected from the hospital records. Their use of health care services, related to the diagnostic process, was gathered. Incremental cost-effectiveness ratio (ICER) per additional diagnosis was calculated from the health care provider perspective. Bootstrapping methods were used to estimate the uncertainty of cost-effectiveness outcomes. Results WES provided a better diagnostic yield (38%) than diagnostic pathway that did not prioritize WES in early diagnosis (25%). WES outperformed other diagnostic paths especially when made early, within one year of first admission (44%). Cost-effectiveness in our results are conservative, affected by WES costs during 2016–18. Conclusions WES is an efficient and cost-effective diagnostic tool that should be prioritized in early diagnostic path of children with progressive neurological disorders. The progressively decreasing price of the test improves cost-effectiveness further.
... Some pilot data on limited numbers of participants has suggested that, as costs reduce, an increased diagnostic yield from genetics and reducing target therapy could indeed become cost effective in clinical practice with obvious differences in diagnostic yield depending on disease process (Sagoo et al., 2017). Whole exome sequencing has already been demonstrated to be more cost effective than traditional diagnostics in paediatric populations with muscle disorders (Schofield et al., 2017), as well as in paediatric monogenetic disorders, particularly when the test is conducted early (Stark et al., 2017). However, mixed success of cost-effective models in adults, such as genetic testing to guide ibrutinib therapy, has demonstrated improved health outcomes but at a significant cost. ...
Article
Full-text available
Since the first polygenic risk score (PRS) in 2007, research in this area has progressed significantly. The increasing number of SNPs that have been identified by large scale GWAS analyses has fuelled the development of a myriad of PRSs for a wide variety of diseases and, more recently, to PRSs that potentially identify differential response to specific drugs. PRSs constitute a composite genomic biomarker and potential applications for PRSs in clinical practice encompass risk prediction and disease screening, early diagnosis, prognostication, and drug stratification to improve efficacy or reduce adverse drug reactions. Nevertheless, to our knowledge, no PRSs have yet been adopted into routine clinical practice. Beyond the technical considerations of PRS development, the major challenges that face PRSs include demonstrating clinical utility and circumnavigating the implementation of novel genomic technologies at scale into stretched healthcare systems. In this review, we discuss progress in developing disease susceptibility PRSs across multiple medical specialties, development of pharmacogenomic PRSs, and future directions for the field.
... In the era of resource and budget constraints, the evaluation of economic implications of providing WES and WGS within clinical settings has a principal role in informing efficient and effective healthcare resource allocation. Despite the high unit costs of WES and WGS, studies have demonstrated the cost-effectiveness of WES and WGS across clinical settings(68)(69)(70)(71)(72). On the other hand, health-economic evidence of rWES and rWGS is rather limited, with the fact that parallel comparison of rWES/rWGS and conventional diagnostic methods is more challenging due to the critical and urgent clinical setting that requires immediate clinical management decisions. ...
Article
Full-text available
The genomics revolution over the past three decades has led to great strides in rare disease (RD) research, which presents a major shift in global policy landscape. While RDs are individually rare, there are common challenges and unmet medical and social needs experienced by the RD population globally. The various disabilities arising from RDs as well as diagnostic and treatment uncertainty were demonstrated to have detrimental influence on the health, psychosocial, and economic aspects of RD families. Despite the collective large number of patients and families affected by RDs internationally, the general lack of public awareness and expertise constraints have neglected and marginalized the RD population in health systems and in health- and social-care policies. The current Coronavirus Disease of 2019 (COVID-19) pandemic has exposed the long-standing and fundamental challenges of the RD population, and has reminded us of the critical need of addressing the systemic inequalities and widespread disparities across populations and jurisdictions. Owing to the commonality in goals between RD movements and universal health coverage targets, the United Nations (UN) has highlighted the importance of recognizing RDs in policies, and has recently adopted the UN Resolution to promote greater integration of RDs in the UN agenda, advancing UN's commitment in achieving the 2030 Sustainable Development Goals of “leav[ing] no one behind.” Governments have also started to launch Genome Projects in their respective jurisdictions, aiming to integrate genomic medicine into mainstream healthcare. In this paper, we review the challenges experienced by the RD population, the establishment and adoption of RD policies, and the state of evidence in addressing these challenges from a global perspective. The Hong Kong Genome Project was illustrated as a case study to highlight the role of Genome Projects in enhancing clinical application of genomic medicine for personalized medicine and in improving equity of access and return in global genomics. Through reviewing what has been achieved to date, this paper will provide future directions as RD emerges as a global public health priority, in hopes of moving a step toward a more equitable and inclusive community for the RD population in times of pandemics and beyond.
... Further evaluation of the cost effectiveness of WGS compared with conventional genetic testing methods (e.g., targeted mtDNA variant, single gene, or gene panel analysis) will be important because the benefits of a single diagnostic blood test to inform directed genetic testing in an extensive number of family members are considerable regarding time to diagnosis and the costs of testing. 53 Comprehensive simultaneous sequencing of both mitochondrial and nuclear genomes by WGS from blood is an accurate and minimally-invasive test to diagnose patients with MDs, avoids tissue biopsies, and has the capability to transform the MD diagnostic pathway. Improvements in health outcomes from early genetic diagnosis, appropriate intervention and treatment, avoidance of adverse events, reduced costs of inappropriate therapy, and potential to prevent disease inheritance are all advantages that could be enabled by the introduction of our WGS analysis pipeline and emphasizes the benefits for integrating WGS into future clinical practice. ...
Article
Full-text available
Objectives Mitochondrial diseases are the commonest group of heritable metabolic disorders. Phenotypic diversity can make molecular diagnosis challenging and causative genetic mutations may reside in either mitochondrial or nuclear DNA. A single comprehensive genetic diagnostic test would be highly useful and transform the field. We applied whole genome sequencing to evaluate the variant detection rate and diagnostic capacity of this technology with a view to simplifying and improving the mitochondrial disease diagnostic pathway. Methods Adult patients presenting to a specialist mitochondrial disease clinic in Sydney, Australia were recruited to the study if they satisfied clinical mitochondrial disease (Nijmegen) criteria. Whole genome sequencing was performed on blood DNA, followed by clinical genetic analysis for known pathogenic mitochondrial disease-associated variants and mitochondrial mimics. Results Of the 242 consecutive patients recruited, 62 subjects had ‘definite’, 108 had ‘probable’ and 72 had ‘possible’ mitochondrial disease classification by the Nijmegen criteria. Disease causing variants were identified for 130 subjects, regardless of the location of the causative genetic mutations, giving an overall diagnostic rate of 53.7% (130/242). Identification of causative genetic mutations informed precise treatment, restored reproductive confidence and optimised patient management. Conclusion Comprehensive bigenomic sequencing accurately detects causative gene mutations in affected patients and simplifies mitochondrial disease diagnosis, enables early treatment and informs the risk of genetic transmission.
... The bars display data from multiple studies ("n" is the total number of individuals) showing the range in diagnostic yields in light gray and the mean with a red line. [1][2][3][4][5][6][7][8][9] other technologies are superior for detecting certain types of CNVs. ...
Article
Full-text available
Exome sequencing (ES) in the clinical setting of inborn metabolic disease (IMDs) has created tremendous improvement in achieving an accurate and timely molecular diagnosis for a greater number of patients, but it still leaves the majority of patients without a diagnosis. In parallel, (personalized) treatment strategies are increasingly available, but this requires the availability of a molecular diagnosis. IMDs comprise an expanding field with the ongoing identification of novel disease genes and the recognition of multiple inheritance patterns, mosaicism, variable penetrance and expressivity for known disease genes. The analysis of trio ES is preferred over singleton ES as information on the allelic origin (paternal, maternal, ‘de novo’) reduces the number of variants that require interpretation. All ES data and interpretation strategies should be exploited including CNV and mitochondrial DNA analysis. The constant advancements in available techniques and knowledge necessitates the close exchange of clinicians and molecular geneticists about genotypes and phenotypes, as well as knowledge of the challenges and pitfalls of ES to initiate proper further diagnostic steps. Functional analyses (transcriptomics, proteomics, metabolomics) can be applied to characterize and validate the impact of identified variants, or to guide the genomic search for a diagnosis in unsolved cases. Future diagnostic techniques (genome sequencing (GS), optical genome mapping, long‐read sequencing, epigenetic profiling) will further enhance the diagnostic yield. We provide an overview of the challenges and limitations inherent to ES followed by an outline of solutions and a clinical checklist, focused on establishing a diagnosis to eventually achieve (personalized) treatment.
... The diagnostic yield for CMD/CM was consistent with previous studies on larger samples utilizing NGS technologies [45,46]. RYR1-related disorders and NEM2, which were the most frequent diagnosis for CMD/CM, are also one of the most prevalent forms of CM in other populations [46,47]. ...
Article
Genetic testing is being considered the first-step in the investigation of hereditary myopathies. However, the performance of the different testing approaches is little known. The aims of the present study were to evaluate the diagnostic yield of a next-generation sequencing panel comprising 39 genes as the first-tier test for genetic myopathies diagnosis and to characterize clinical and molecular findings of families from southern Brazil. Fifty-one consecutive index cases with clinical suspicion of genetic myopathies were recruited from October 2014 to March 2018 in a cross-sectional study. The overall diagnostic yield of the next-generation sequencing panel was 52.9%, increasing to 60.8% when including cases with candidate variants. Multi-gene panel solved the diagnosis of 12/25 (48%) probands with limb-girdle muscular dystrophies, of 7/14 (50%) with congenital muscular diseases, and of 7/10 (70%) with muscular dystrophy with prominent joint contractures. The most frequent diagnosis for limb-girdle muscular dystrophies were LGMD2A/LGMD-R1-calpain3-related and LGMD2B/LGMD-R2-dysferlin-related; for congenital muscular diseases, RYR1-related-disorders; and for muscular dystrophy with prominent joint contractures, Emery-Dreifuss-muscular-dystrophy-type-1 and COL6A1-related-disorders. In summary, the customized next-generation sequencing panel when applied in the initial investigation of genetic myopathies results in high diagnostic yield, likely reducing patient's diagnostic odyssey and providing important information for genetic counseling and participation in disease-specific clinical trials.
Article
Genetic condition is one of the major etiologies causing morbidity and mortality in infants and children. More and more etiologies can be solved using next-generation sequencing (NGS) as it develops. Currently, whole-exome sequencing (WES) and whole-genome sequencing (WGS) have been highly integrated into clinical practice. The average diagnostic yield of WES/WGS in pediatric patients with genetic condition was around 40% (range: 21%–80%), with acceptable turnaround time and cost. The higher diagnostic yield categories are deafness, ophthalmic, neurological, skeletal conditions, and inborn error of metabolism. Positive results provide benefit with those for actionable diseases, next pregnancy planning, and family members. For those in critical condition, with the emergence of sequencing technology and bioinformatics analysis tools, provisional diagnosis can be made as short as 13.5 h using ultrarapid WGS. We believe this powerful tool has changed pediatric daily practice.
Article
Background Hereditary neuromuscular diseases (NMDs) are a group of rare disorders, and the diagnosis of these diseases is a substantial burden for referral centers. Although next-generation sequencing (NGS) has identified a large number of genes associated with hereditary NMDs, the diagnostic rates still vary across centers. Methods Patients with a suspected hereditary NMD were referred to neuromuscular specialists at the National Taiwan University Hospital. Molecular diagnoses were performed by employing a capture panel containing 194 genes associated with NMDs. Results Among the 50 patients referred, 43 had a suspicion of myopathy, and seven had polyneuropathy. The overall diagnostic rate was 58%. Pathogenic variants in 19 genes were observed; the most frequent pathogenic variant found in this cohort (DYSF) was observed in only four patients, and 10 pathogenic variants were observed in one patient each. One case of motor neuron disease was clinically mistaken for myopathy. A positive family history increased the diagnostic rate (positive: 72.7% vs. negative: 56.3%). Fourteen patients with elevated plasma creatine kinase levels remained without a diagnosis. Conclusion The application of NGS in this single-center study proves the great diversity of hereditary NMDs. A capture panel is essential, but high-quality clinical and laboratory evaluations of patients are also indispensable.
Article
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Objective Next Generation Sequencing (NGS) is increasingly used for the diagnosis of rare genetic disorders. The aim of this study is to review the different approaches for economic evaluations of Next Generation Sequencing (NGS) in pediatric care used to date, to identify all costs, effects, and time horizons taken into account. Methods A systematic literature review was conducted to identify published economic evaluations of NGS applications in pediatric diagnostics, i.e. exome sequencing (ES) and/or genome sequencing (GS). Information regarding methodological approach, costs, effects, and time horizon was abstracted from these publications. Results Twenty-eight economic evaluations of ES/GS within pediatrics were identified. Costs included were mainly restricted to direct in-hospital healthcare costs and varied widely in inclusion of sort of costs and time-horizon. Nineteen studies included diagnostic yield and eight studies included cost-effectiveness as outcome measures. Studies varied greatly in terms of included sort of costs data, effects, and time horizon. Conclusion Large differences in inclusion of cost and effect parameters were identified between studies. Validity of outcomes can therefore be questioned, which hinders valid comparison and widespread generalization of conclusions. In addition to current health economic guidance, specific guidance for evaluations in pediatric care is therefore necessary to improve the validity of outcomes and furthermore facilitate comparable decision-making for implementing novel NGS-based diagnostic modalities in pediatric genetics and beyond.
Article
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Background: Fetal akinesia/hypokinesia, arthrogryposis and severe congenital myopathies are heterogeneous conditions usually presenting before or at birth. Although numerous causative genes have been identified for each of these disease groups, in many cases a specific genetic diagnosis remains elusive. Due to the emergence of next generation sequencing, virtually the entire coding region of an individual's DNA can now be analysed through "whole" exome sequencing, enabling almost all known and novel disease genes to be investigated for disorders such as these. Methods: Genomic DNA samples from 45 patients with fetal akinesia/hypokinesia, arthrogryposis or severe congenital myopathies from 38 unrelated families were subjected to next generation sequencing. Clinical features and diagnoses for each patient were supplied by referring clinicians. Genomic DNA was used for either whole exome sequencing or a custom-designed neuromuscular sub-exomic supercapture array containing 277 genes responsible for various neuromuscular diseases. Candidate disease-causing variants were investigated and confirmed using Sanger sequencing. Some of the cases within this cohort study have been published previously as separate studies. Results: A conclusive genetic diagnosis was achieved for 18 of the 38 families. Within this cohort, mutations were found in eight previously known neuromuscular disease genes (CHRND, CHNRG, ECEL1, GBE1, MTM1, MYH3, NEB and RYR1) and four novel neuromuscular disease genes were identified and have been published as separate reports (GPR126, KLHL40, KLHL41 and SPEG). In addition, novel mutations were identified in CHRND, KLHL40, NEB and RYR1. Autosomal dominant, autosomal recessive, X-linked, and de novo modes of inheritance were observed. Conclusions: By using next generation sequencing on a cohort of 38 unrelated families with fetal akinesia/hypokinesia, arthrogryposis, or severe congenital myopathy we therefore obtained a genetic diagnosis for 47 % of families. This study highlights the power and capacity of next generation sequencing (i) to determine the aetiology of genetically heterogeneous neuromuscular diseases, (ii) to identify novel disease genes in small pedigrees or isolated cases and (iii) to refine the interplay between genetic diagnosis and clinical evaluation and management.
Article
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Objective: To establish and evaluate the effectiveness of a comprehensive next-generation sequencing (NGS) approach to simultaneously analyze all genes known to be responsible for the most clinically and genetically heterogeneous neuromuscular diseases (NMDs) involving spinal motoneurons, neuromuscular junctions, nerves, and muscles. Methods: All coding exons and at least 20 bp of flanking intronic sequences of 236 genes causing NMDs were enriched by using SeqCap EZ solution-based capture and enrichment method followed by massively parallel sequencing on Illumina HiSeq2000. Results: The target gene capture/deep sequencing provides an average coverage of ∼1,000× per nucleotide. Thirty-five unrelated NMD families (38 patients) with clinical and/or muscle pathologic diagnoses but without identified causative genetic defects were analyzed. Deleterious mutations were found in 29 families (83%). Definitive causative mutations were identified in 21 families (60%) and likely diagnoses were established in 8 families (23%). Six families were left without diagnosis due to uncertainty in phenotype/genotype correlation and/or unidentified causative genes. Using this comprehensive panel, we not only identified mutations in expected genes but also expanded phenotype/genotype among different subcategories of NMDs. Conclusions: Target gene capture/deep sequencing approach can greatly improve the genetic diagnosis of NMDs. This study demonstrated the power of NGS in confirming and expanding clinical phenotypes/genotypes of the extremely heterogeneous NMDs. Confirmed molecular diagnoses of NMDs can assist in genetic counseling and carrier detection as well as guide therapeutic options for treatable disorders.
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Glycosylphosphatidylinositol (GPI) anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (∼20-50% compared to controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This paper contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data. © The Author 2015. Published by Oxford University Press.
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Neuromuscular disorders are a clinically, pathologically, and genetically heterogeneous group. Even for the experienced clinician, an accurate diagnosis is often challenging due to the complexity of these disorders. Here, we investigated the utility of next generation sequencing (NGS) in early diagnostic algorithms to improve the diagnosis for patients currently lacking precise molecular characterisation, particularly for hereditary myopathies. 43 patients presenting with early onset neuromuscular disorders from unknown genetic origin were tested by NGS for 579 nuclear genes associated with myopathy. In 21 of the 43 patients, we identified the definite genetic causes (48.8%). Additionally, likely pathogenic variants were identified in seven cases and variants of uncertain significance (VUS) were suspected in four cases. In total, 19 novel and 15 known pathogenic variants in 17 genes were identified in 32 patients. Collagen VI related myopathy was the most prevalent type in our cohort. The utility of NGS was highlighted in three cases with congenital myasthenia syndrome, as early diagnosis is important for effective treatment. A targeted NGS can offer cost effective, safe and fairly rapid turnaround time, which can improve quality of care for patients with early onset myopathies and muscular dystrophies; in particular, collagen VI related myopathy and congenital myasthenia syndromes. Nevertheless, a substantial number of patients remained without molecular diagnosis in our cohort. This may be due to the intrinsic limitation of detection for some types of mutations by NGS or to the fact that other causative genes for neuromuscular disorders are yet to be identified. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
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Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.
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Over the past decade there have been major advances in defining the genetic basis of the majority of congenital myopathy subtypes. However the relationship between each congenital myopathy, defined on histological grounds, and the genetic cause is complex. Many of the congenital myopathies are due to mutations in more than one gene, and mutations in the same gene can cause different muscle pathologies. The International Standard of Care Committee for Congenital Myopathies performed a literature review and consulted a group of experts in the field to develop a summary of 1. the key features common to all forms of congenital myopathy and 2. the specific features that help to discriminate between the different genetic subtypes. The consensus statement was refined by two rounds of on-line survey, and a three-day workshop. This consensus statement provides guidelines to the physician assessing the infant or child with hypotonia and weakness. We summarise the clinical features that are most suggestive of a congenital myopathy, the major differential diagnoses and the features on clinical examination, investigations, muscle pathology and muscle imaging that are suggestive of a specific genetic diagnosis to assist in prioritisation of genetic testing of known genes. As next generation sequencing becomes increasingly used as a diagnostic tool in clinical practice, these guidelines will assist in determining which sequence variations are likely to be pathogenic.
Article
OBJECTIVES: To evaluate the diagnostic outcomes in a large cohort of congenital muscular dystrophy (CMD) patients using traditional and Next Generation Sequencing (NGS) technologies. METHODS: 123 CMD patients were investigated using the traditional approaches of histology, immunohistochemical analysis of muscle biopsy and candidate gene sequencing. Undiagnosed patients available for further testing were investigated using NGS. RESULTS: Muscle biopsy and immunohistochemical analysis found deficiencies of laminin α2, α-dystroglycan or collagen VI in 50% of patients. Candidate gene sequencing and chromosomal microarray established a genetic diagnosis in 32% (39/123). Of 85 patients presenting in the last 20 years, 28 of 51 who lacked a confirmed genetic diagnosis (55%) consented to NGS studies, leading to confirmed diagnoses in a further 11 patients. Using the combination of approaches, a confirmed genetic diagnosis was achieved in 51% (43/85). The diagnoses within the cohort were heterogeneous. 45/59 probands with confirmed or probable diagnoses had variants in genes known to cause CMD (76%), and 11/59 (19%) had variants in genes associated with congenital myopathies, reflecting overlapping features of these conditions. One patient had a congenital myasthenic syndrome and two had microdeletions. Within the cohort, five patients had variants in novel (PIGY and GMPPB) or recently published genes (GFPT1 and MICU1) and seven had variants in TTN or RYR1; large genes that are technically difficult to Sanger sequence. INTERPRETATION: These data support NGS as a first-line tool for genetic evaluation of patients with a clinical phenotype suggestive of CMD, with muscle biopsy reserved as a second-tier investigation. This article is protected by copyright. All rights reserved.
Article
Objectives: To evaluate the diagnostic outcomes in a large cohort of congenital muscular dystrophy (CMD) patients using traditional and Next Generation Sequencing (NGS) technologies. Methods: 123 CMD patients were investigated using the traditional approaches of histology, immunohistochemical analysis of muscle biopsy and candidate gene sequencing. Undiagnosed patients available for further testing were investigated using NGS. Results: Muscle biopsy and immunohistochemical analysis found deficiencies of laminin α2, α-dystroglycan or collagen VI in 50% of patients. Candidate gene sequencing and chromosomal microarray established a genetic diagnosis in 32% (39/123). Of 85 patients presenting in the last 20 years, 28 of 51 who lacked a confirmed genetic diagnosis (55%) consented to NGS studies, leading to confirmed diagnoses in a further 11 patients. Using the combination of approaches, a confirmed genetic diagnosis was achieved in 51% (43/85). The diagnoses within the cohort were heterogeneous. 45/59 probands with confirmed or probable diagnoses had variants in genes known to cause CMD (76%), and 11/59 (19%) had variants in genes associated with congenital myopathies, reflecting overlapping features of these conditions. One patient had a congenital myasthenic syndrome and two had microdeletions. Within the cohort, five patients had variants in novel (PIGY and GMPPB) or recently published genes (GFPT1 and MICU1) and seven had variants in TTN or RYR1; large genes that are technically difficult to Sanger sequence. Interpretation: These data support NGS as a first-line tool for genetic evaluation of patients with a clinical phenotype suggestive of CMD, with muscle biopsy reserved as a second-tier investigation. This article is protected by copyright. All rights reserved.
Article
Importance To our knowledge, the efficacy of transferring next-generation sequencing from a research setting to neuromuscular clinics has never been evaluated.Objective To translate whole-exome sequencing (WES) to clinical practice for the genetic diagnosis of a large cohort of patients with limb-girdle muscular dystrophy (LGMD) for whom protein-based analyses and targeted Sanger sequencing failed to identify the genetic cause of their disorder.Design, Setting, and Participants We performed WES on 60 families with LGMDs (100 exomes). Data analysis was performed between January 6 and December 19, 2014, using the xBrowse bioinformatics interface (Broad Institute). Patients with LGMD were ascertained retrospectively through the Institute for Neuroscience and Muscle Research Biospecimen Bank between 2006 and 2014. Enrolled patients had been extensively investigated via protein studies and candidate gene sequencing and remained undiagnosed. Patients presented with more than 2 years of muscle weakness and with dystrophic or myopathic changes present in muscle biopsy specimens.Main Outcomes and Measures The diagnostic rate of LGMD in Australia and the relative frequencies of the different LGMD subtypes. Our central goals were to improve the genetic diagnosis of LGMD, investigate whether the WES platform provides adequate coverage of known LGMD-related genes, and identify new LGMD-related genes.Results With WES, we identified likely pathogenic mutations in known myopathy genes for 27 of 60 families. Twelve families had mutations in known LGMD-related genes. However, 15 families had variants in disease-related genes not typically associated with LGMD, highlighting the clinical overlap between LGMD and other myopathies. Common causes of phenotypic overlap were due to mutations in congenital muscular dystrophy–related genes (4 families) and collagen myopathy–related genes (4 families). Less common myopathies included metabolic myopathy (2 families), congenital myasthenic syndrome (DOK7), congenital myopathy (ACTA1), tubular aggregate myopathy (STIM1), myofibrillar myopathy (FLNC), and mutation of CHD7, usually associated with the CHARGE syndrome. Inclusion of family members increased the diagnostic efficacy of WES, with a diagnostic rate of 60% for “trios” (an affected proband with both parents) vs 40% for single probands. A follow-up screening of patients whose conditions were undiagnosed on a targeted neuromuscular disease–related gene panel did not improve our diagnostic yield.Conclusions and Relevance With WES, we achieved a diagnostic success rate of 45.0% in our difficult-to-diagnose cohort of patients with LGMD. We expand the clinical phenotypes associated with known myopathy genes, and we stress the importance of accurate clinical examination and histopathological results for interpretation of WES, with many diagnoses requiring follow-up review and ancillary investigations of biopsy specimens or serum samples.
Article
The statistic of interest in the economic evaluation of health care interventions is the incremental cost effectiveness ratio (ICER), which is defined as the difference in cost between two treatment interventions over the difference in their effect. Where patient-specific data on costs and health outcomes are available, it is natural to attempt to quantify uncertainty in the estimated ICER using confidence intervals. Recent articles have focused on parametric methods for constructing confidence intervals. In this paper, we describe the construction of non-parametric bootstrap confidence intervals. The advantage of such intervals is that they do not depend on parametric assumptions of the sampling distribution of the ICER. We present a detailed description of the non-parametric bootstrap applied to data from a clinical trial, in order to demonstrate the strengths and weaknesses of the approach. By examining the bootstrap confidence limits successively as the number of bootstrap replications increases, we conclude that percentile bootstrap confidence interval methods provide a promising approach to estimating the uncertainty of ICER point estimates. However, successive bootstrap estimates of bias and standard error suggests that these may be unstable; accordingly, we strongly recommend a cautious interpretation of such estimates. © 1997 John Wiley & Sons, Ltd.