28 03

Technology Evaluation Center
Special Report: Exome

Sequencing for Clinical
Diagnosis of Patients with
Suspected Genetic Disorders
Assessment
Program
Volume 28, No. 3
August 2013
Executive Summary
Background
There are different clinical diagnostic scenarios for which traditional Sanger DNA sequencing technology is clinically available. Its success as a diagnostic tool depends on the accuracy and completeness of the differential diagnosis, as well as the number and locations of mutations responsible for the
observed disease. Single-gene disorders are diseases that result from a mutation in one gene that has
a large effect on phenotype. A clinical focus of this Special Report is the scenario of an undiagnosed,
suspected single-gene disorder that features multiple congenital anomalies. Disorders (often early
developmental) that present with multiple anomalies suggest a genetic etiology. They are often clinically difficult to diagnose due to nonspecific presentation, potentially overlapping differential diagnoses (clinical heterogeneity) or a rare disorder, and lack of a clear diagnostic testing path. The search
for a clinical diagnosis may result in a long (months to years) diagnostic odyssey comprising several
specialist consults and many different individual molecular, cytogenetic and biochemical tests, as well
as other types of diagnostic procedures. A relatively unbiased sequencing application that examines
most genes without requiring prior knowledge of potential diagnoses would be advantageous.
Single-gene disorders are inherited in various patterns (e.g., autosomal dominant, autosomal recessive, X-linked) and are affected by penetrance (proportion of individuals with the disease-related
mutation who have the disease phenotype at all) and by expressivity (variable phenotypes that can
occur in different individuals with the same genetic mutation). Disorders arising from a mutation in a
single gene may be caused by exactly the same mutation in all individuals with the condition, or may
be caused by any one of many different mutations in the same gene (allelic heterogeneity). In contrast, a large proportion of heritable disorders are genetically heterogeneous; clinically indistinguishable or overlapping phenotypes may be caused by mutations in different genes (locus heterogeneity).
The number of genes involved can range from just a few to hundreds. For many of these genetically
heterogeneous disorders, the full complement of causal genes is not yet known.
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Determining genetic causality for disease and establishing a molecular diagnosis in clinical practice
is challenging; in recent years, molecular technologies have strikingly increased gene discovery and
understanding the causes of single-gene disorders among patients with suspected, but previously
undiagnosed, genetic disorders. The value of molecular diagnosis in these patients can be viewed
from a clinical perspective similar to other diagnostic tests; it is also commonly addressed from
perspectives of clinical and personal utility, as well as a societal view. A molecular diagnosis can:
confirm a suspected or established clinical diagnosis; inform prognosis; aid in selecting treatment,
surveillance or preventive options; reveal mode of inheritance; identify carrier/risk status of family
members; and guide research regarding new therapy or patient management. Available guidelines
are currently focused on molecular diagnosis, distinguishing among phenotypically overlapping possible conditions, and on referral for genetic consultation.
NOTICE OF PURPOSE: TEC Assessments are scientific opinions, provided solely for informational purposes. TEC Assessments
should not be construed to suggest that the Blue Cross Blue Shield Association, Kaiser Permanente Medical Care Program or the
TEC Program recommends, advocates, requires, encourages, or discourages any particular treatment, procedure, or service; any
particular course of treatment, procedure, or service; or the payment or non-payment of the technology or technologies evaluated.
2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited.
Sanger sequencing is currently the gold standard for molecular diagnosis of unknown point
mutations in known genes (usually limited to the coding regions and intron/exon splice sites).
However, when the gene in question is very large or when there is substantial locus heterogeneity,
comprehensive Sanger sequencing may become unacceptably burdensome. For rare conditions,
this may preclude laboratories from offering such testing, consequently making genetic testing
unavailable for diagnostic purposes.
Recently, next-generation sequencing technologies have become more accessible in terms of cost,
analytic validity, and rapidity. Exome sequencing has the capacity to determine in a single assay
an individuals exomic variation profile, limited to most of the protein coding sequence of an
individual (approximately 85%), composed of about 20,000 genes, 180,000 exons (protein-coding
segments of a gene), and constituting approximately 1% of the whole genome. It is believed that
the exome contains about 85% of heritable disease-causing mutations. In 2009, the first proof-ofprinciple study was published in which the authors developed an exome sequencing method and
applied it to samples from unrelated individuals all known to have the same Mendelian disorder,
for which the gene was already established. Mutations in the known gene were identified as
uniquely common to all patients. Less than 3 years later, a MEDLINE search on Mendelian
disorders using exome sequencing identified publications on over 100 diseases and more than
100 newly identified genes.
Exome sequencing has the advantages of speed and efficiency relative to Sanger sequencing of
multiple genes, delivering results in weeks to months. After candidate variants can be sufficiently
reduced in number (from an initial 2030,000 per individual exome), targeted Sanger sequencing
may be used to confirm the presence or absence of the potential variant(s) in the individual being
diagnosed, and in affected and unaffected family members. In this way it is relatively straightforward to perform exome sequencing as a clinical service for new patients to help determine a
clinical diagnosis and identify causal variants in known genes.
Exome sequencing, relying on next-generation sequencing technologies, is not without challenges
and limitations. In the human exome, about 1 in every 1,000 nucleotides will vary. Computer
algorithms can eliminate most that are presumed irrelevant to the condition of concern. The final
steps of variant filtering involve manual research and review. This requires expertise including knowledge of the patients phenotype and family history, extremely current knowledge and
exploration of published variant-disease associations, and judgment, all of which can contribute
to variability in the final variant interpretation. Reproducibility at the level of final manual variant
interpretation has not been fully characterized; comparisons of exome capture platforms using
detection of known medically relevant variants as the sequencing result for assessment indicate
small differences in detection, gaps in capture platforms, as well as regions of poor coverage that
are unlikely to benefit from improved capture or greater depth of sequencing. Tools to improve
the computational and variant analysis pipeline are available and under development.
Like Sanger sequencing, next-generation (Next-Gen or NGS) sequencing cannot detect large
deletions or duplications of DNA or nucleotide repeats that can cause disease. Error rates due to
uneven sequencing coverage, gaps in exon capture prior to sequencing, difficulties with narrowing the large initial number of variants to manageable numbers without losing likely candidate
mutations, poorly annotated variant databases, lack of standardized procedures, and identifying
mutations in unrelated genes or unknown genes are all issues. Detailed guidance from regulatory
or professional organizations is under development, and the variability contributed by the different platforms and procedures used by clinical laboratories offering exome sequencing as a clinical
service is unknown. There are also ethical questions about reporting incidental findings, such
as identifying medically relevant mutations in genes unrelated to the diagnostic question, sex
chromosome abnormalities and non-paternity when family studies are performed.
Exome Sequencing for Clinical Diagnosis of Patients with Suspected Genetic Disorders
Objective
The objective of this Special Report is to compare Sanger sequencing and next-generation
sequencing clinical molecular methods for diagnosing disorders that are caused by mutations in a
single gene, and the potential uses of exome sequencing for molecular diagnosis. We first describe
exome sequencing using next-generation sequencing and compare it with Sanger sequencing.
Second, three clinical scenarios caused by single-gene disorders where exome sequencing might
be applied are described. Finally, we review evidence related to its clinical use in evaluating
patients with undiagnosed suspected genetic disorders accompanied by multiple anomalies
focusing on diagnostic yield and potential impact on patient outcomes.
Search Strategy
MEDLINE was searched via PubMed using the search string (Sequence Analysis, DNA[MeSH]
OR ((DNA OR gene) AND (sequence analysis OR sequencing))) AND ((Mutation[MeSH]) OR
(Polymorphism, Single Nucleotide[MeSH] OR Polymorphism, Genetic[MeSH] ) OR mutation*
OR polymorphism*) AND (Exome[MeSH] OR exom*), through March 2013. A search performed
July 2013 identified no studies that would affect conclusions.
Selection Criteria
Studies of clinical diagnosis included for review met the following criteria:
n Patients were described as having an undiagnosed suspected genetic disorder
accompanied by multiple anomalies
n Studies employed exome sequencing by next-generation technology
n Purpose of study appeared to be focused on patient(s) and on improving diagnostic
accuracy and possible therapy, or on molecular method as a way to improve diagnosis
n Patients selected for exome sequencing were primary, not secondary to purpose of study
n Actual disorder investigated was secondary to purpose of study
n Conclusions focused on patients and at least on improved methods of clinical
diagnosis (if not on treatment outcomes)
Main Results
The diagnostic yield of exome sequencing in the 6 larger patient series (n>10; each study
sequenced 12 to 118 exomes) varied from 10% to 54%. Results were more often successful in
the 21 small individual/family studies (n10; each study sequenced 1 to 10 exomes). Since these
studies were largely positive or negative on the basis of the index case, and few negative results
were found in this group of studies, selective reporting of positive results could have occurred.
Beyond diagnostic yield, occasional anecdotal reports were identified of clinical benefit following molecular diagnosis by exome sequencing. Such potential benefits included a revision of the
original diagnosis; a few instances of treatments initiated or discontinued as a result of the known
functional consequences of the mutation, and genetic counseling to the affected patient and/or
family members. However, no systematic study of clinical outcomes was identified.
Studies of exome sequencing compared with traditional Sanger sequencing generally agree
that exome sequencing is more efficient and more likely to result in a diagnosis. One study
reported a sensitivity of 98.3% for detecting previously identified mutations, as well as benign
variants. Particular limitations noted were gaps in coverage of the exome and difficulty interpreting variants.
Authors Conclusions and Comment
Exome sequencing using next-generation technology has recently become available as a laboratory-developed diagnostic clinical laboratory test. A major indication for use is molecular diagnosis of
patients with suspected genetic disorders. These patients may be left without a satisfactory clinical
diagnosis of their disorder despite a lengthy diagnostic odyssey involving a variety of traditional
molecular and other conventional diagnostic tests. For some patients, exome sequencing obtained
after initial diagnostic evaluation (that may include other genetic testing) has failed may avoid
the diagnostic odyssey and return a likely causal variant. Currently, the diagnostic yield appears
2013 Blue Cross and Blue Shield Association. Reproduction without prior authorization is prohibited. 3
to be no greater than 50% and possibly less for patients with suspected genetic disorder accompanied by multiple anomalies. Medical management decisions, including initiation of new treatment or discontinuing inappropriate treatment, may result for only a subset of those diagnosed.
Reproductive decisions for parents considering an additional pregnancy may be informed by
determining the mode of inheritance. Appropriate use of exome sequencing requires considerable
genetic, clinical, and genetic counseling expertise.
Contents
Objective5
Discussion24
Background5
Conclusions25
Methods17
References27
Review17
Published in cooperation with Kaiser Foundation Health Plan and

Southern California Permanente Medical Group.
TEC Staff Contributors
Lead AuthorMargaret Piper, Ph.D., M.P.H.; Co-AuthorMark D. Grant M.D., M.P.H.; TEC Executive DirectorNaomi Aronson,
Ph.D.; TEC Director, Technology AssessmentsMark D. Grant, M.D., M.P.H.; Director, Clinical Science Services
Kathleen M. Ziegler, Pharm.D.; Research/Editorial StaffClaudia J. Bonnell, B.S.N., M.L.S.; Kimberly L. Hines, M.S.
AcknowledgmentsStaff would like to acknowledge the contributions of Scott McLean, M.D., to the research and development
of this Special Report.
Blue Cross and Blue Shield Association Medical Advisory Panel
Trent T. Haywood, M.D., J.D.Chairman, Senior Vice President, Clinical Affairs/Medical Director, Blue Cross and Blue Shield
Association; Steven N. Goodman, M.D., M.H.S., Ph.D.Scientific Advisor, Dean for Clinical and Translational Research, Stanford
University School of Medicine, Professor, Departments of Medicine, Health Research and Policy; Mark A. Hlatky, M.D.Scientific
Advisor, Professor of Health Research and Policy and of Medicine (Cardiovascular Medicine), Stanford University School of Medicine.
Panel Members Peter C. Albertsen, M.D., Professor, Chief of Urology, and Residency Program Director, University of
Connecticut Health Center; Sarah T. Corley, M.D., F.A.C.P., Chief Medical Officer, NexGen Healthcare Information Systems,
Inc.American College of Physicians Appointee; Helen Darling, M.A., President, National Business Group on Health;
Josef E. Fischer, M.D., F.A.C.S., William V. McDermott Professor of Surgery, Harvard Medical SchoolAmerican College
of Surgeons Appointee; I. Craig Henderson, M.D., Adjunct Professor of Medicine, University of California, San Francisco;
Jo Carol Hiatt, M.D., M.B.A., F.A.C.S., Chair, Inter-Regional New Technology Committee, Kaiser Permanente; SairaA.Jan,
M.S., Pharm.D., Associate Clinical Professor, Ernest Mario School of Pharmacy, Rutgers, The State University
of New Jersey, Residency Director and Director of Clinical Programs Pharmacy Management, Horizon Blue Cross and
Blue Shield of New Jersey; Thomas Kowalski, R.Ph., Clinical Pharmacy Director, Blue Cross Blue Shield of Massachusetts;
Lawrence Hong Lee, M.D., M.B.A., F.A.C.P., Vice President and Executive Medical Director for Quality and Provider Relations,
Blue Cross and Blue Shield of Minnesota; Bernard Lo, M.D., Professor of Medicine and Director, Program in Medical
Ethics, University of California, San Francisco; Randall E. Marcus, M.D., Charles H. Herndon Professor and Chairman,
Department of Orthopaedic Surgery, Case Western Reserve University School of Medicine; Barbara J. McNeil, M.D., Ph.D., Ridley
Watts Professor and Head of Health Care Policy, Harvard Medical School, Professor of Radiology, Brigham and Womens Hospital;
William R. Phillips, M.D., M.P.H., Clinical Professor of Family Medicine, University of WashingtonAmerican Academy
of Family Physicians Appointee; Richard Rainey, M.D., Medical Director, Regence BlueShield of Idaho; Rita F. Redberg,
M.D., M.Sc., F.A.C.C., Professor of Medicine and Director, Womens Cardiovascular Services, University of California San Francisco;
Maren T. Scheuner, M.D., M.P.H., F.A.C.M.G., Chief, Medical Genetics, VA Greater Los Angeles Healthcare System; Associate
Clinical Professor, Department of Medicine, David Geffen School of Medicine at UCLA, Affiliate Natural Scientist, RAND Corporation;
J. Sanford Schwartz, M.D., F.A.C.P., Leon Hess Professor of Medicine and Health Management & Economics, School of Medicine and
The Wharton School, University of Pennsylvania.
CONFIDENTIAL: This document contains proprietary information that is intended solely for Blue Cross and Blue Shield Plans
and other subscribers to the TEC Program. The contents of this document are not to be provided in any manner to any other
parties without the express written consent of the Blue Cross and Blue Shield Association.
Objective
The objective of this Special Report is to
compare Sanger sequencing and next-generation sequencing clinical molecular methods for
diagnosing disorders that are caused by mutations in a single gene, and the potential uses
of exome sequencing for molecular diagnosis.
We first describe exome sequencing using
next-generation sequencing and compare it
with Sanger sequencing. Second, three clinical scenarios caused by single-gene disorders
where exome sequencing might be useful are
described. Finally, we review evidence related
to use of exome sequencing in evaluating
patients with undiagnosed suspected genetic
disorders accompanied by multiple anomaliesfocusing on diagnostic yield and potential
impact on patient outcomes.
Background
Definitions and Epidemiology
Single-gene disorders are the result of a mutation in one gene that has a large effect on
phenotype. Mutations may be inherited or
may arise de novo in parental gametes during
meiosis.1 Such disorders are often classified
as rare or orphan diseases because they affect
only small populations. There is no universal
definition of a rare disease; a 1984 amendment
to the 1983 Orphan Drug Act defines a rare
disease as a disease or condition that affects
fewer than 200,000 people in the U.S. This
definition was repeated in the Rare Disease
Act of 2002 that established the (already existing) National Institutes of Health Office of Rare
Disease Research in statute. The number of
rare diseases has been estimated at 5,000 to
8,000 (IOM, 2010), and is rapidly growing. The
prevalence of specific single-gene disorders
may vary among different populations that
are defined ethnically and geographically.
Collectively, an estimated 25 to 30 million
people in the United States are believed to have
a rare disease (NIH Office of Rare Diseases
Research, http://rarediseases.info.nih.gov/
AboutUs.aspx).
Single-gene disorders may be expressed in

utero, early during postnatal development, or
across the childhood and adult age spectrum.
We consider different clinical diagnostic scenarios for which traditional, Sanger sequencing
technology is already clinically available, but
its success as a diagnostic tool depends on the
accuracy and completeness of the differential
diagnosis (e.g., skill and experience of ordering clinician). Clinical scenarios where nextgeneration sequencing might be particularly
useful include 2:
1. A suspected single-gene disorder that
features multiple congenital anomalies.
Disorders (often early developmental) that
present with an unusual constellation of
findings or multiple congenital anomalies, suggest a genetic etiology, and may be
exceedingly difficult to diagnose clinically
due to nonspecific presentation or potentially
overlapping differential diagnoses (clinical heterogeneity). The search for a clinical
diagnosis may result in a long (potentially
months to years) diagnostic odyssey comprising several specialist consults and many
different individual molecular, cytogenetic
and biochemical tests, as well as other types
of diagnostic procedures. For example, the
Undiagnosed Diseases Program at the
National Institutes of Health enrolls and
evaluates patients with conditions that have
long eluded diagnosis (http://rarediseases.
info.nih.gov/Resources.aspx?PageID=31).
Arriving at a molecular diagnosis may
inform management decisions, determine
risk for family members, and can provide
information for reproductive decision making of the patient or a patients parents.
2. A known single-gene disorder with locus
heterogeneity and the need for subtype
identification. A mutation in one of several
different genes may cause the disorder.
Molecular diagnostic testing is not usually
necessary for diagnosis but may be useful in
some cases for confirmation or for identifying subtypes of the disorder with different
symptoms and requiring different management decisions. It may also determine risk
The presence of a mutation found in an individual but not the parents may also be due to post-conception mutations that have
attained a high level of somatic mosaicism (i.e., a mixture of cells with and without the new mutation). This happens either
because the mutations occurred at an early stage of embryonic development or because the cells with the variant had better
survival or increased proliferation.
2
In addition to these scenarios where traditional sequencing is available, there are others where exome sequencing may also
be used for a suspected single-gene disorder where Sanger sequencing is not available because the responsible gene is not known
or suspected.
1
for family members and provide helpful

information for reproductive decisionmaking. An example is congenital disorders of
glycosylation (CDG), a large group of inherited metabolic disorders affecting several
steps of the protein glycosylation pathway.
There are numerous subtypes depending
on the affected gene. Subtypes have been
described in only a few individuals, limiting understanding of the phenotype, and
the range of phenotypic expression across
subtypes is broad. For some subtypes, the
differential diagnosis may overlap with other
disorders such as mitochondrial disease
or congenital muscular dystrophies. The
diagnostic test for all subtypes of CDG is
transferrin isoform analysis; however, while
the specific enzymes are known, enzymatic
assays are not generally available. To identify specific subtypes, molecular diagnosis is
necessary. Traditional diagnostic sequencing of each of the known associated genes is
available to identify variants for most subtypes and may be helpful for those few subtypes where treatment other than supportive
treatment is available to alleviate symptoms.
The most likely subtypes must be prioritized
by clinical indications and sequencing tests
selected accordingly.
3. A suspected single-gene disorder that
features a common disease (e.g., cancer)
with locus heterogeneity. Knowledge of
the genetic diagnosis may influence clinical
management, including decisions regarding
surveillance and prevention; may determine
risk for family members; and may provide
helpful information for reproductive decisionmaking. An example is a patient with
a personal and family history of early onset
breast cancer. This scenario may be encountered with an inherited mutation in one of
several known genes (e.g., BRCA1, BRCA2,
PALB2, PTEN, TP53, STK11, CHEK2, ATM)
or unknown genes. Sanger sequencing can
evaluate mutations in each of these genes
one by one. This typically begins with the
BRCA1/2 genes due to the relatively higher
prevalence of mutations in these genes.
However, because not all genes associated
with inherited breast cancer have been identified, some families may exhaust the known
list of associated genes without identifying
a family mutation.
As suggested by the examples, traditional
sequencing can be highly informative, but
6
frequently is limited in scope and requires

knowing the most likely diagnosis and the
genes associated with that diagnosis. An unbiased sequencing application that examines
the exome without requiring prior knowledge
would offer some advantages in all three scenarios. The use of next-generation sequencing
to identify potential causative variants in the
exome is an unbiased sequencing application
that does not require prior clinical knowledge
of the diagnosis or of the genes contributing
to the diagnosis to direct test selection. This
Special Report addresses these scenarios while
focusing on evidence for clinical utility in the
first clinical scenario, i.e., suspected singlegene disorders accompanied by multiple congenital anomalies.
Patterns of Inheritance. The inheritance patterns of single-gene disorders resemble those
originally described by Mendel:
n
Inherited disorders are autosomal if they

are encoded by genes on one of the 22 pairs
of autosomes (non-sex chromosomes).
n Inherited disorders are X-linked if encoded
by a gene on the X chromosome.
n Dominant conditions are those expressed
in heterozygotes, i.e., individuals with one
copy of a mutant allele and one copy of a
normal (wild-type) allele.
n Recessive conditions are clinically manifest
only in individuals homozygous for the
mutant allele (or individuals said to be
compound heterozygotes because they
carry two different mutant alleles), i.e.,
carrying a double dose of an abnormal
gene, or hemizygous for an X-linked
recessive condition.
A non-Mendelian inheritance pattern is that
of mitochondrial inheritance. Mitochondria
are normal cellular organelles located in the
cytoplasm and are involved in energy production. Mitochondrial genes are inherited only
from the mother because they are contained
in the cytoplasm of the ovum.
The presence of causative gene variants that
are not found in the parents can be due to
de novo mutations that occurred in the parental
gametes during meiosis. Such mutations are
then heritable only if the individual with the
disorder has children. Other explanations for
mutations not found in the parents include nonpaternity and parental germline mosaicism.
Inheritance patterns can sometimes be determined by analyzing the family history or

pedigree, i.e., tracking the individuals who do
and do not express disease within the context
of their family relationships. Discerning such
patterns is important for making a genetic diagnosis, genetic counseling for reproductive risks,
carrier testing, and identifying at-risk relatives
of an individual with disease. However, inheritance patterns can be complicated by reduced
penetrance (not all individuals with the diseaserelated mutation will develop a disease phenotype) and by variable expressivity (variable
phenotypes that can occur in different individuals with the same genetic mutation).
Genetic Heterogeneity. Disorders known to
be caused by mutations in a single gene may
be caused by exactly the same mutation in all
individuals with the condition. For example,
sickle cell disease, an autosomal recessive
disorder, is caused by a specific and invariant
point mutation in the gene for the beta-globin
chain of hemoglobin, resulting in the amino
acid glutamic acid to be replaced with valine
at the sixth position (Lazarus and Schmaier,
2011). Cystic fibrosis, however, can be caused
by any one of over 1,500 mutations in the gene
coding for the cystic fibrosis transmembrane
conductance regulator (CFTR) protein; this is
allelic heterogeneity (Bobadilla et al. 2002).
Cystic fibrosis is also an autosomal recessive
disorder and can result from the same mutation
in each copy of the CFTR gene (homozygous
alleles) or different mutations in each gene
copy (compound heterozygote). The various
mutations have a range of effects on the CFTR
protein resulting in a spectrum of mild to
severe disease.
fewer than 5 disorders had known molecular

genetic causes (Ginsburg 2011). By 1990 the
number had increased to about 150, and by
2011 was over 3,000. Since then new, confirmed
gene-disease associations (and descriptions
of new syndromes with molecular diagnoses)
have been published at a rapid rate. However,
for many single-gene developmental disorders
a causative gene remains to be discovered.
The value of molecular diagnosis in patients
with undiagnosed suspected genetic disorders
can be considered from a clinical perspective,
similar to other diagnostic tests, but is also
commonly addressed from a patient, family,
and societal perspective. The following are
possible uses of molecular diagnosis; not all
apply to every single-gene disorder, particularly
where treatment is not yet available. Anecdotal
examples can be found for each, but the
overall contribution of each type of outcome
to the total picture of heritable disorders is
not available.
n
n
n
n
n
Confirm a suspected or clinically

established genetic disease diagnosis
Determine prognosis
Aid in selecting patient treatment,
and risk-appropriate surveillance and
prevention options
Determine carrier/risk status
of family members
Determine reproductive risk for the patient
with a genetic diagnosis, or for couples
with an affected child who are considering
an additional pregnancy
Guide research regarding new therapy
or patient management
In contrast, a large proportion of single-gene

disorders are genetically heterogeneous: clinically indistinguishable or overlapping phenotypes may be caused by mutations in different
genes (also termed locus heterogeneity).
The number of associated genes can range
from just a few to hundreds. For many of these
genetically heterogeneous disorders, the full
complement of causal genes is unknown.
As an example, Miller et al. (2011) note that

for patients with Charcot-Marie-Tooth (CMT)
disease, which to date is associated with causative mutations in over 50 genes/loci accounting for only a portion of affected patients,
genetic testing may be performed for several
possible reasons: identifying the inheritance
pattern of their CMT, making family planning
decisions, and obtaining knowledge about the
cause and natural history of their form of CMT.
Molecular Diagnosis. Given the complexity

of genetic diseases, determining causation for
disease and establishing a molecular diagnosis
in clinical practice is challenging. The contribution of modern genomic technologies to gene
discovery and molecular diagnosis of singlegene disorders has been phenomenal. In 1982,
Several guidelines address molecular diagnosis,

in the context of the genetic testing methods
available at the time the guidelines were
written and published; Table 1 lists examples
(note that all but the American College of
Medical Genetics and Genomics guideline
address targeted testing for specific disorders
or syndromes). Consistent themes are that

molecular diagnosis is important for classifying
disorders and distinguishing among overlapping conditions; in some cases is necessary for
confirming a diagnosis; and supplies important
information for genetic counseling.
When a disease-causing gene(s) is established,
assays based, for example, on polymerase
chain reaction technology can be designed to
specifically detect known mutations for clinical
diagnosis. When many different point mutations in a gene are possible, Sanger sequencing, the current gold standard for detecting
unknown point mutations, can be employed to
determine the entire sequence of the coding
and intron/exon splice sites of gene regions
where mutations are most likely to be found.
However, when genes are large and mutations
are possible in many or all exons (proteincoding regions of the gene), and when there
is genetic (locus) heterogeneity, comprehensive
Sanger sequencing may be prohibitively laborious and costly.
Currently available clinical assays designed for
molecular diagnosis may be incomplete due
to genetic heterogeneity and unknown causative genes, or because only a portion of the
known genes and mutations can be efficiently
tested using conventional molecular methods.
Recently, next-generation sequencing technologies have become more accessible in terms of
cost and speed; it is a technology that has been
adopted by a growing number of molecular
genetic clinical laboratories.
Next-generation sequencing platforms
produced by different companies use different technologies, but in general are high
throughput, producing thousands or millions
of sequences at the same time. These methods
offer increased sequencing capacity while
decreasing some of the labor and cost associated with Sanger sequencing. However, time
and costs for interpretation are increased. Nextgeneration sequencing can be used to sequence
the genome of an individual, or selected portions of the genome (e.g., a targeted panel that
includes all genes known to be related to a
particular condition or group of disorders such
as the inherited cardiomyopathies; this eliminates the challenge of dealing with mutations
found in unknown genes). Genome sequencing
has the capacity to determine an individuals
complete genomic variation profile in a single
experiment (Gilissen et al. 2011). However,
8
genome sequencing is currently limited in

terms of throughput, data analysis, and cost
efficiency, although rapid improvements continue.
Exome Sequencing. Exome sequencing
consists of similar methods as genome sequencing, except for an extra step involving exome
capture, which limits sequencing to the protein
coding regions of the genome, composed of about
20,000 genes, 180,000 exons (protein-coding
segments of a gene), and constituting approximately 1% of the whole genome. It is believed
that the exome contains about 85% of heritable
disease-causing mutations (Choi et al. 2009).
Prior to determining the sequence of an
individuals exome, the exome must first be
physically captured from the sample by
various available methods. Capture designs
usually include all protein coding sequences,
flanking splice sequences, and untranslated
regions that could contain damaging variants.
Different sources (e.g. the National Center for
Biotechnology Information Consensus Coding
DNA Sequence) may be used as the reference
gene set for the design template. Thus, there
is variability across capture methods (Fuchs
et al. 2012).
After an individuals exome is sequenced, a
search for the disease-causing mutation is
performed by comparing the sequencing data
with a human genome reference, resulting in
a list of all non-reference variants. Typically,
2030,000 variants result for each exome
sequence (Robinson et al. 2011). Variants are
first screened according to various parameters, e.g., depth of coverage (a minimum of
10 sequencing reads per base are needed for
a basic level of reliability of the variant call;
recommended numbers of reads are typically
much higher and vary depending on the specific application). The next step is to distinguish
between normal variation in the general population and possible disease-causing variants
using a variety of publicly available databases.
Variants predicted to be benign are computationally removed. Once the variant list is sufficiently reduced to a relatively small number,
the remaining variants can be combined with
information known about the disease to determine if the list includes potential variants that
are known or suspected to be deleterious (e.g.,
nonsense or frameshift mutations) and that
explain the patient phenotype.
Table 1. Examples of Guidelines/Policies that Address Molecular Testing in Hereditary Disorders
Year
Focus of Guideline
Impact of Genetic Testing
American College
of Medical Genetics
and Genomics
(ACMG, 2012)
2012
Clinical applications of
genomic sequencing
Exome sequencing should be considered for clinical diagnosis

of an affected individual when:
a genetic etiology is likely but targeted testing is not available
patients disorder has high genetic heterogeneity
targeted testing resulted in no diagnosis
a fetus has a likely genetic disorder and targeted tests are
negative; turnaround time should be considered
Policy specifically references exome

sequencing (and also whole genome
sequencing for same indications)
American Academy
of Neurology
(Ashwal et al. 2004)
2004
Diagnostic assessment
of the child with cerebral
palsy
Differentiate between cerebral palsy and other neurometabolic

disorders that may share symptomology during early years;
some non-cerebral palsy disorders may benefit from early
diagnosis and available treatment (e.g., glutaric aciduria)
None specified
American Academy
of Neurology
(Ashwal et al. 2009)
2009
Evaluation of the child

with microcephaly
Microcephaly has been associated with numerous genetic

etiologies; genetic etiologies have been reported in 15 to
53% of cases using targeted testing; currently available data
likely underestimate the contribution of genetic testing to the
diagnostic evaluation
Targeted testing may be considered

to determine etiology; as the yield
of screening tests (not including
exome sequencing) is unknown, no
recommendations can be made
European Federation of
Neurological Societies
(Gasser et al. 2010)
2010
Molecular diagnosis
of ataxias and spastic
paraplegias
Hereditary ataxias must be distinguished from non-hereditary

forms; different forms have different modes of inheritance
and are clinically overlapping, making them hard to diagnose
without molecular testing; hereditary spastic paraplegia
syndromes are also clinically heterogeneous and are classified
based on mode of inheritance and genetic linkage
Targeted testing for specific syndromes,

in some cases depending on clinical
presentation and the results of other
laboratory tests
(Burgunder et al. 2010)
2010
Molecular diagnosis
of channelopathies,
epilepsies, migraine,
stroke and dementias
Channelopathies: molecular testing may aid diagnosis but

cannot be considered routine due to the large number of
genes and mutations;
Cerebrovascular diseases: targeted tests suggested to help
resolve specific clinical situations;
Familial hemiplegic migraine: confirm diagnosis by sequencing
most commonly associated gene;
Alzheimers disease: useful for genetic counseling in early
onset autosomal dominant form of disease

as recommended in clinical context
Type of Genetic Testing/

Algorithm Referenced
Guideline Source
Guideline Source
Year
Focus of Guideline
Impact of Genetic Testing
Type of Genetic Testing/

Algorithm Referenced
(Albanese et al. 2011)
2011
Diagnosis and treatment

of primary dystonias
Diagnosis is clinical; genetic testing can aid in classification,

which is important for appropriate management, prognostic
information, and genetic counseling
Targeted testing for some specific forms

of dystonia is available, as recommended
in clinical context
(Finsterer et al. 2009)
2009
Molecular diagnosis of
mitochondrial disorders
Diagnostic workup follows a stepwise procedure:

1) initiate comprehensive family history and clinical review;
2) determine syndromic vs. non-syndromic form and inheritance
pattern; 3) results of 1 and 2 determine recommended genetic
tests for likely syndromic forms
Various types of genetic tests depending

on differential diagnosis; many genes/
types of mutations are possible
(Bushby et al. 2010)
2010
Diagnosis and
management of Duchenne
muscular dystrophy
Mutation testing in the DMD gene is necessary to confirm

diagnosis, even when absence of dystrophin protein expression
on muscle biopsy has been shown; results provide information
required for genetic counselling, prenatal diagnosis, and
consideration for future mutation-specific therapies
Deletion/duplication analysis followed by

gene sequencing if negative; many and
different types of mutations are possible
(Norwood et al. 2007)
2007
Diagnosis and
management of limb girdle
muscular dystrophies
Detection of an associated mutation is the standard of

diagnosis and necessary to be able to offer carrier or presymptomatic testing to other family members Mutation
detection for the rarer types of LGMD may only be available
on a research basis. May change counseling advice e.g.,
if differential includes Becker muscular dystrophy

per clinical differential diagnosis
American Academy
of Neurology
(England et al. 2009)
2009
Evaluation of distal
symmetric polyneuropathy
Genetic testing should be conducted for the accurate diagnosis

and classification of hereditary neuropathies the genetic
testing profile should be guided by the clinical phenotype,
inheritance pattern, and electrodiagnostic (EDX) features
Targeted testing per decision algorithm
10
Table 1. Examples of Guidelines/Policies that Address Molecular Testing in Hereditary Disorders (contd)
In 2009, Ng et al. (2009) published a proof of

principle study in which the authors developed
next-generation sequencing methods for targeted sequencing of all protein-coding regions
of the genome (i.e., the exome). Exome
sequencing was applied to samples from 4
unrelated individuals known to have FreemanSheldon syndrome, for which the associated
gene was already known. The method identified variants in the known gene as uniquely
common to the 4 patients. A second proof of
principle study (Choi et al. 2009) used exome
sequencing to identify a variant in the SLC26A3
gene, known to be associated with congenital
chloride diarrhea, in an infant originally suspected to have a renal defect such as Bartter
syndrome. Targeted sequencing of the gene
in additional patients with suspect Bartter
syndrome but no related gene variants found
SLC26A3 variants and the diagnoses were subsequently revised to congenital chloride diarrhea, illustrating the unbiased nature of exome
sequencing investigations.
Exome sequencing has the advantages of speed
and efficiency relative to Sanger sequencing,
delivering results in weeks to months. If candidate variants can be sufficiently reduced in
number, targeted Sanger sequencing may be
used to confirm the presence or absence of
the potential variant(s) in the individual being
diagnosed. In addition, the variant call may be
influenced by how the variant segregates in
affected and unaffected family members (e.g.,
if a heterozygous mutation is not found in either
parent, the variant is assumed de novo and more
likely causative). In this way it is relatively
straightforward to perform exome sequencing
as a clinical service for patients to determine
a molecular diagnosis by identifying causal
variants in known genes (Lyon and Wang,
2012). Table 2 compares Sanger and nextgeneration sequencing in different clinical
settings showing some of the major advantages
and disadvantages of each. Table 3 reviews
limitations of both methods.
If no likely causal variants in genes known to
be associated with the condition are identified,
some clinical laboratories offer gene discovery
as a clinical service, requiring exome sequencing of additional family members. The variant
list for the affected individual can be compared with sequencing information from key
individuals from the same family. For example,

in the case of an autosomal recessive disorder,
the affected individual must be homozygous or
compound heterozygous for the causal variants,
each of which occurs only once in each parent.
For autosomal dominant conditions, if neither
parent carries the mutation and there is no evidence of non-paternity, then there is evidence
for a de novo mutation that is more likely deleterious. In this way, candidate gene variants
can be rapidly reduced to one or a few candidates for further investigation. The number of
patients needed to resolve potential causative
variants depends on the pattern of inheritance.
For example, because the number of possible
causative variants is much higher in autosomal
dominant disorders, more family members may
be needed to determine co-segregation of a
variant with the phenotype (Fuchs et al. 2012).
Limitations of Exome Sequencing. Similar to
Sanger sequencing, exome sequencing is not
without limitations. For example, causative
variants that occur in non-coding regulatory
regions are not detected (Lyon and Wang, 2012)
and certain types of mutations are not detected
(large deletions, duplications, rearrangements,
nucleotide repeats, and epigenetic changes).
Exome capture methods may not capture each
exon in every gene and insufficient depth of
coverage in some regions can mask a diseasecausing variant (McDonald et al. 2012). Due to
these and other detection limitations (see Table
3), the total proportion of gene variants that
will be missed by current methods may be as
high as 15-20% (Fuchs et al. 2012). Additionally,
public databases containing information on
putative disease-causing mutations are incomplete and may have high error rates (Bell et al.
2011) requiring manual curation; associations
for some mutations in the database may not
be causal. Improvements in shared databases
are ongoing.
Finally, next-generation sequencing has not
yet been reviewed by the U.S. Food and Drug
Administration, and only general technical
guidelines have been discussed by a few organizations (Gargis et al. 2012; Schrijver et al.
2012). The College of American Pathologists
Laboratory Accreditation Program has revised
their molecular pathology checklist to include a
dedicated section on next-generation sequencing as of July 31, 20123. Several laboratories
See CAP press release available at http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fpor

tlets%2FcontentViewer%2Fshow&_windowLabel=cntvwrPtlt&cntvwrPtlt%7BactionForm.contentReference%7D=media_
resources%2Fnewsrel_checklist_next_gene.html&_state=maximized&_pageLabel=cntvwr
Sequencing Technology
Sanger
Next Generation
Gold standard for detecting unknown point

mutations in coding and exon/exon splice sites
Yes
Not yet
Maximum feasible size of clinical sequencing unit
Single-gene exon(s), single gene (if not excessively large)
Exome (whole genome likely, in future)
Ability to identify a previously established causal
Relatively efficient; if a small number of known mutations

account for all known disease association, if there are
common, ancestry-specific mutations, or if there is a
known familial mutation
Inefficient for most circumstances of a known causal variant;

single-gene assays in development e.g. for cystic fibrosis
Requires accurate and complete differential diagnosis

with knowledge of causative genes. Genes known to be
associated with the disorder are sequenced one at a time
in order of likelihood of harboring a mutation, based
on prevalence and clinical findings. Currently available
clinical tests may not address all mutations or all genes
known to be associated with heterogeneous disorders,
and clinical tests may not be available for known genes.
The exome or a panel of genes known to be associated with the

disorder can all be done in a single sequencing assay; public
databases help filter variants and identify previously established
causal variants. Targeted sequencing of family members for
variant may be needed in follow-up; if known variant not
identified, may provide initial steps in discovery of new variant
or new gene-disease association. If gene panel is performed,
then unknown genes will be missed.
variant in a single gene
Ability to identify a causal variant for a suspected

single-gene disorder that features a common
disease with locus heterogeneity, need for
subtype identification, or identify a causal variant
Unknown genes will be missed.
12
Table 2. Comparison of Standard Sanger Sequencing vs. Next-generation Sequencing
Sequencing Technology
Major limitations
(see also Limitations of exome sequencing,
following)
Sanger
Next Generation
Sequence may contain gaps due to biases in cloning

amplification step
Sequence likely to contain gaps due to uneven efficiency of

exome capture and amplification
Structural variation (e.g. large deletions, duplications

and rearrangements) not detected
Structural variation (e.g. large deletions, duplications and

rearrangements) not detected
Nucleotide repeat disorders not detected
Nucleotide repeat disorders not detected
Epigenetic changes not detected
Epigenetic changes not detected
One or a few variants of uncertain significance

may be detected
Thousands of variants of uncertain significance will be detected
Reduced efficiency/cost-effectiveness when full sequences

of large single genes or full or partial sequences of
several genes are required; Sanger sequencing of full
exome cost prohibitive
More efficient/cost effective when sequences of several genes or

of whole exome are required; and when several patients batched
Identifying deleterious variants (mutations) in unknown genes
(i.e., genes that have not yet been characterized)
Incidental findings of medically relevant/actionable variants; sex
chromosomal abnormalities; and non-paternity if family studies
are performed to interpret variants
Table 2. Comparison of Standard Sanger Sequencing vs. Next-generation Sequencing (contd)
Change in DNA
Explanation
Some exons
Some exons are missed by current capture methods
Structural variants
Structural variants, such as chromosomal translocations and inversions that relocate or reverse a section of DNA but
dont otherwise alter the sequence are not detected. Large deletions, duplications and rearrangements of DNA within
genes will not be detected (e.g., most cases of Duchenne/Becker muscular dystrophy).
Triplet repeat sequences
Sequences of base triplet repeats, as are found in most hereditary ataxia syndromes, Huntington and fragile X
syndrome/fragile X-associated tremor ataxia syndrome, are not detected or quantitated
Copy number variants
Othercopy number variantsinvolving entire genes or partial gene deletions and insertions presently are not detectable
Intronic sequences in genes
Splice site mutations that cause missing exons will not be detected
Uniparental disomy
In uniparental disomy, 2 mutations are inherited from one parent, rather than one from each. Determination of risk
for future siblings is different in each case; however, this distinction cannot be made in an exome screen.
Non-coding regulatory sequence mutations
Much of the human genome is composed of regulatory regions that are not part of the exome. Mutations in regulatory
regions (e.g., promoter) can critically affect protein production in a particular gene or metabolic pathway.
Gene-gene interactions
One gene affecting the expression of another can explain differing levels of expression of the same single-gene
disease. Computational tools are needed to describe networks of interacting genes revealed in exome sequencing.
Epigenetic changes
Non-sequence-related modifications to DNA, such as the addition of methyl groups can profoundly affect gene
expression. Exome sequencing does not detect such modifications.
14
Table 3. Types of DNA Changes Not Detected by Exome Sequencing
offer diagnostic exome sequencing (Table 4).

Platforms and procedures are likely to vary
from lab to lab; any consequent variability in
sequencing results is unknown.
The reproducibility of exome sequencing
has not been fully characterized at the
level of final manual variant interpretation.
Performance in a laboratory licensed under
the Clinical Laboratory Improvement Act
(CLIA) for high-complexity testing requires
delivery of a reproducible set of called,
quality-filtered variants from the sequencing platform. CLIA requirements attempt to
minimize false positives with increased depth
of sequencing. The quality calculation occurs
prior to variant annotation, filtering, and
manual interpretation for patient diagnosis.
Several reports have compared commercially
available exome capture platforms, which
can introduce variability into the sequencing
assay. These reports have used various types
of sequencing results to assess comparability
and reproducibility, including medically relevant single nucleotide polymorphism
(SNP) variant detection.
Asan et al. (2011) found that three exome
capture platforms showed similar high power
at 30 sequencing depth in interrogating
known medically interesting rare SNPs filtered
to remove variants present in the general
population; small differences reflected the
variations in design of each platform. Clark et
al. and Sulonen et al. also compared exome
capture platforms and concluded that all platforms could detect disease-associated variants
but that a small proportion was unique to each
platform (Clark et al. 2011; Sulonen et al. 2011).
In comparing two exome capture platforms,
Parla et al. (2011) determined that the genotypic information resulting from exome capture
and sequencing provides sensitivities up to
97%, false discovery rates up to 0.67% for all
variants and up to approximately 1.5% for
heterozygous variants.
The final steps of variant filtering involve
manual curation, requiring expertise, knowledge of the patients phenotype and family
history, extremely current knowledge and
exploration of published variant-disease
associations, and judgment, all of which can
contribute to variability in the final variant
interpretation. New tools such as VAR-MD generate a ranked list of variants using predicted
pathogenicity, Mendelian inheritance models,

genotype quality, and population variant frequency data to standardize and automate
much of the manual process (Sincan et al.
2012). Integrated networks such as Galaxy
provide a framework for tracking, recording
and disseminating all details of computational analyses conducted during an exome
sequence evaluation (Nekrutenko and Taylor,
2012). However, sufficiently detailed guidelines
and possibly regulatory standards are needed
to advise the best use of such tools within the
overall sequencing pipeline and to ensure comparable quality and reproducibility of exome
sequencing results across laboratories.
Finally, there are ethical questions about
reporting incidental or off-target findings. For
example, exome sequencing may be ordered to
aid in diagnosis of a developmental syndrome
in a young child; in addition to answering
the diagnostic question, a medically relevant
variant such as a BRCA1/2 gene mutation may
be discovered, which predicts a very high
risk of breast cancer and ovarian cancer at
an adult age. Should all potentially relevant
variants, only medically actionable variants
with known clinical utility, or only the results
addressing the original clinical question that
prompted the test order be reported to the
physician and patient? Opinions differ considerably and various groups are studying the
issue. On March 21, 2013 the American College
of Medical Genetics and Genomics (ACMG)
released Recommendations for Reporting
of Incidental Findings in Clinical Exome and
Genome Sequencing (available at http://www.
acmg.net//AM/Template.cfm?Section=Home3)
in which a list of genes and classes or types of
mutations for each is provided. The ACMG
recommend that laboratories performing clinical exome/genome sequencing seek and report
the mutations listed. Other challenges include
the finding of sex chromosome abnormalities
(e.g., Klinefelter and Turner syndrome), and if
family studies are performed to inform variant
classification, non-paternity may be revealed.
Also challenging is whether or how to follow-up
an identified potentially deleterious mutation
in a gene not previously associated with the
condition.
Summary. There are many clinical, genetic,
and genetic testing complexities that must
be taken into account prior to obtaining
exome sequencing. Appropriate use requires
Laboratory
Laboratory Indications for Testing
Ambry Genetics, Aliso Viejo, CA

http://ambrygen.com/exome-sequencing
The patients clinical presentation is unclear/atypical disease and there are multiple genetic
conditions in the differential diagnosis.
GeneDx, Gaithersburg, MD
http://www.genedx.com/test-catalog/xomedx/
?gclid=CNWCjdyt8LQCFcxAMgodhVUA4A
a patient with a diagnosis that suggests the involvement of one or more of many different
genes, which would, if even available and sequenced individually, be prohibitively expensive
Baylor College of Medicine, Houston, TX

https://www.bcm.edu/geneticlabs/test_detail.cfm?testcode=1500&show=1
used when a patients medical history and physical exam findings strongly suggest that there is
an underling genetic etiology. In some cases, the patient may have had an extensive evaluation
consisting of multiple genetic tests, without identifying an etiology
University of California Los Angeles Health System:

http://pathology.ucla.edu/body.cfm?id=393
This test is intended for use in conjunction with the clinical presentation and other markers of
disease progression for the management of patients with rare genetic disorders.
EdgeBio, Gaithersburg, MD
http://www.edgebio.com/clinical-partner-program
Recommended In situations where there has been a diagnostic failure with no discernible
path In situations where there are currently no available tests to determine the status of a
potential genetic disease In situations with atypical findings indicative of multiple disease[s]
Childrens Mercy Hospitals and Clinics, Kansas City

http://www.childrensmercy.org/Health_Care_Professionals/Research/
Pediatric_Genomic_Medicine/Exome_Sequencing/
Provided as a service to families with children who have had an extensive negative work-up
for a genetic disease; also used to identify novel disease genes.
Emory Genetics Laboratory, Atlanta, GA

http://genetics.emory.edu/egl/tests/?testid=4162
Indicated when there is a suspicion of a genetic etiology contributing to the probands

manifestations
16
Table 4. Examples of Laboratories Offering Exome Sequencing as a Clinical Service
substantial clinical and genetic expertise.

Figure 1 summarizes the general role exome
sequencing can play in genetic diagnosis (it
does not incorporate all the necessary considerations to address before ordering for an individual patient).
FDA Status. No exome sequencing assays
have been submitted to or reviewed by the
U.S.Food and Drug Administration (FDA).
Clinical laboratories may develop and validate
tests in-house (laboratory-developed tests or
LDTs; previously called home-brew) and
market them as a laboratory service; LDTs
must meet the general regulatory standards
of the Clinical Laboratory Improvement Act
(CLIA). Laboratories offering LDTs must be
licensed by CLIA for high-complexity testing.
Methods
Search Methods
MEDLINE was searched via PubMed using
the search string Sequence Analysis, DNA
[MeSH]) AND Mutation[MeSH] AND exom*
resulting in 265 entries on January 18, 2013.
Using this search, a partial version of Table 5
was compiled, and Key Terms from the studies
included were compared to construct a broader
search strategy.
A second search string, (Sequence Analysis,
DNA[MeSH] OR ((DNA OR gene) AND
(sequence analysis OR sequencing))) AND
((Mutation[MeSH]) OR (Polymorphism,
Single Nucleotide[MeSH] OR Polymorphism,
Genetic[MeSH]) OR mutation* OR polymorphism*) AND (Exome[ MeSH] OR exom*),
through March 2013. These were screened for
studies to be included in the complete version
of Table 5. A search performed July 2013
identified no studies that would affect conclusions and the original set of included studies is
reported here.
The National Guidelines Clearinghouse on
genetic diagnosis (unlinked), resulting in 202
entries on January 24, 2013. These results were
screened for relevant guidelines regarding
molecular diagnosis in heritable disorders to
include in Table 1 (Background).
Study Selection
Studies included for review met the following
criteria4:
n Patients were described as having an undiagnosed suspected genetic disorder accompanied by multiple anomalies
n Studies employed exome sequencing by
massively parallel sequencing technology
n Purpose of study appeared to be focused
on patient(s) and on improving diagnosis and possible therapy, or on molecular
method as a way to improve diagnosis
n Patients selected for exome sequencing were
primary, not secondary to purpose of study
n Actual disorder investigated was secondary
to purpose of study
n Conclusions focused on patients and at least
on improved methods of clinical diagnosis
(if not on treatment outcomes)
Medical Advisory Panel Review
This Special Report was reviewed by the Blue
Cross and Blue Shield Association Medical
Advisory Panel (MAP) on June 6, 2013. In order
to maintain the timeliness of the scientific
information in this Special Report, literature
searches were performed subsequent to the
Panels review. If the search updates identified
any additional studies that met the criteria for
detailed review, the results of these studies
were included in the tables and text where
appropriate. There were no studies that would
change the conclusions of this Special Report.
Review
The purpose of this portion of this Special
Report is to review studies of diagnostic exome
sequencing in patients with a suspected genetic
disorder accompanied by multiple anomalies,
with particular attention to the reported impact
or potential impact of the exome sequencing
results on patient outcomes as discussed in the
Background section of this Report.
Studies that met the inclusion criteria for
clinical diagnosis studies are summarized in
Table 5 and organized into larger patient series
(n>10), smaller patient/family studies (n10),
and studies of the exome sequencing method.
Note that the table specifies how many affected
individuals had their exome sequenced; in
all studies, additional genetic studies such as
The intent of some of these criteria was to exclude studies of discovery of new gene-disease associations.
Probable Genetic Etiology

Based on Phenotype/History
(exclude microdeletions/
duplications by CMA testing;
exclude inborn errors
of metabolism)
Phenotype
Consistent with Single
Gene Disorder
Yes
No*
Specific Gene
Test Available
Yes
No
Exome Sequencing
No
Treatment or
Interventions
Based on Result
*Applies whether or not genetic heterogeneity likely present
Use Available Test
Test Diagnostic
18
Figure 1. General Algorithm for the Role of Exome Sequencing in Genetic Diagnosis
(see also Points to Consider in the Clinical Application of Genomic Sequencing http://www.acmg.net/StaticContent/PPG/Clinical_Application_of_Genomic_Sequencing.pdf).
As indicated in the text, not all patients will receive a diagnosis from exome sequencing.
Study
Final Diagnosis; Patient(s) Description
Diagnostic
Yield (%)
Results
Patient series, exome sequencing assays n>10

Gahl et al.
2012
29
Summary of the National Institutes of Health Undiagnosed

Diseases Program; details the Programs application of genomic
technology to establish diagnoses, and the Programs 2-year
success rate
10
Exome sequencing contributed to the diagnosis in 3 of 29

affected cases in which exome sequencing was used; patients
accepted into the program remained undiagnosed despite
exhaustive workup
Need et al.
2012
12
Patients with unexplained intellectual disability and/or

developmental delay; one major congenital anomaly; 23 minor
congenital anomalies; and facial dysmorphisms; previously
normal chromosomal microarray
50
Obtained a likely genetic diagnosis in 6 of the 12 probands,

including the identification of apparently causal mutations in 4
genes known to cause Mendelian disease and one gene related
to known Mendelian disease genes
Dixon-Salazar
et al. 2012
118
Probands with pediatric-onset neurodevelopmental disease

(intellectual disability, epilepsy, autism, structural brain diseases,
and neuromuscular disorders), known causes excluded; all born
to consanguineous parents
27
Exome sequencing identified 22 new disease-associated genes;

10 probands had initial diagnoses changed, leading to changes in
management for some
Futema et al.
2012
48
Familial hypercholesterolemia (FH); patients known to have

FH but no gene mutations by standard genetic tests
54%
Exome sequencing identified 17 disease-associated mutations in

LDLR (including 2 novel variants highly likely to be pathogenic)
and 2 in APOB; 2 likely pathogenic LDLR variants and 5 uncertain
APOB variants were also found
(19 mutations
+ 2 variants)
Choi et al.
2012
25
Charcot-Marie-Tooth (CMT) disease; patients with clinically

diagnosed CMT disease but no recognized duplications/deletions
or mutations known to be CMT-associated by available testing
32
Exome sequencing identified causative heterozygous

mutations in 5 different genes in 8 patients; for genetically and
phenotypically heterogeneous genetic disorders like CMT with
more than 50 causative genes/loci, exome sequencing is an
efficient method with respect to accuracy, speed, and cost
Eng et al.
2012 [Meeting
Abstract]
60
Various diagnoses; report of first samples submitted for clinical

testing; patients had complex neurological phenotypes and
extensive prior genetic testing without an etiologic diagnosis
27
Exome sequencing identified some medically actionable variants

including those causing G-6-PD, Marfan syndrome,
and arrhythmogenic right ventricular dysplasia
Table 5. Studies of Clinical Diagnosis Using Exome Sequencing
Study
Diagnostic
Yield (%)
Results
Patient/family studies, exome sequencing assays n10

Johansson
et al. 2012
Patients with suspected maturity-onset diabetes of the young

(MODY)
33
Study sought to improve differential diagnosis among MODY

subtypes in order to determine treatment; resulted in a genetic
diagnosis in 3 of 9 patients; novel variants need further
investigation; improved coverage is needed
Haack et al.
2012
10
Unrelated individuals with mitochondrial complex I deficiency

of childhood onset, a group of highly heterogeneous conditions
affecting 1 in 5 ,000 10,000 live births
70
Known or potentially pathogenic variants were identified in 5

genes in 7 of 10 patients; in at least one case, identification of
the causative variant gene led to corrective treatment (high-dose
riboflavin supplementation in a patient with mutations in ACAD9)
Motazacker
et al. 2012
Autosomal dominant hypercholesterolemia
Leidenroth
et al. 2012
Limb-girdle muscular dystrophy; patient initially diagnosed with

facioscapulohumeral muscular dystrophy
(1/1)
Exome sequencing revised original diagnosis
McDonald
et al. 2012
Limb-girdle muscular dystrophy exhibits genetic and phenotypic

heterogeneity; here, two families with autosomal dominantly
inherited forms were investigated
(5/5)
Causative mutations were identified in 2 families; one gene is

known to be involved in muscle disease pathogenesis; other gene
is related to cellular actin networks; previously associated with
limb-girdle phenotype
Worthey et al.
2011
X-linked inhibitor of apoptosis deficiency; 15-month infant

presented with a severe intractable Crohns disease-like illness
suggesting an immunodeficiency, but comprehensive clinical
evaluation did not result in diagnosis or clear treatment
(1/1)
Although immune reconstitution was already a treatment option,

likelihood of success was unknown without an underlying
mechanism of disease; exome sequencing revealed a mutation in
the X-linked inhibitor of apoptosis gene; patient symptoms were
an unusual expression of this deficiency syndrome; allogeneic
stem-cell transplant was recommended and alleviated GI disease
Haugarvoll
et al. 2013
Alpha-methylacyl-coA racemase (AMACR) deficiency;

Siblings initially suspected of mitochondrial disease were
diagnosed with AMACR deficiency, has a heterogeneous
clinical presentation
(2/2)
Exome sequencing revised initial diagnosis; associated MRI

features were described that may help guide earlier diagnosis
in these patients; one patient was started on a diet restricting
phytanic and pristanic acid intake, which may be beneficial
Improve diagnostic yield; Novel APOB mutations were

unexpectedly found outside the regularly screened region; the
new mutations significantly attenuate binding to the LDL receptor
20
Table 5. Studies of Clinical Diagnosis Using Exome Sequencing (contd)
Diagnostic
Yield (%)
Results
Patient/family studies, exome sequencing assays n10 (contd)

Knies et al.
2012
Fanconi anemia; patients diagnosed by non-molecular procedures

but no known mutations
(4/4)
3 of 4 detected mutations were new in already known diseaseassociated genes; cost and time was variable but lower than
traditional methods would have been; traditional methods would
not have identified all mutations
Mallott et al.
Ataxia telangiectasia (AT); infants screened negative for severe

combined immunodeficiency (SCID) but found to have T
lymphocytopenia were diagnosed with AT
(2/2 and 7 of
an additional
13 AT stored
newborn
samples)
Exome sequencing revised initial diagnosis; AT may be

detectable by SCID screening; exome sequencing may be best
follow-up for large AT gene; early detection permits avoidance
of infectious complications, calculation of recurrence risk for
families and of increased cancer risks for patients and carriers
Kim et al.
2012
Inborn error of vitamin B12 metabolism; child with

hyperhomocysteinemia and methylmalonic aciduria could not be
diagnosed with usual biochemical and genetic tests
(1/1)
Exome sequencing identified only the third known patient with

the cblJ cobalamin cofactor disorder
Tsurusaki et al.
2013
Joubert syndrome (JS) and related disorders, which are

genetically heterogeneous (17 causative genes known to date)
(5/5 families)
Exome sequencing advantageous with regard to time and cost

compared to conventional testing
Hanchard et al.
2013
Hypokalemic periodic paralysis; young child with episodic lower

limb weakness; clinical and laboratory evaluations were equivocal
and standard molecular testing was negative
(1/1)
Exome sequencing revealed a mutation in the calcium channel

gene CACNA1S, previously reported in a single patient as a
cause of an atypical form of hypokalemic periodic paralysis;
results provided additional evidence for long-term potassium
supplementation and allowed counseling regarding a sibling and
regarding recurrence risk
Andrade et al.
2012
Progressive myoclonus epilepsy attributable to neuronal ceroid

lipofuscinosis; 3 family members affected with autosomal
recessive disease, all negative for extensive pathologic and
genetic analyses over 10 years
(1/1)
Exome sequencing replaces extensive and repetitive diagnostic

odyssey; neuronal ceroid lipofuscinosis is a common cause of
cortical neurodegeneration, and results from mutations in genes
CLN1 CLN10
Dauber et al.
2013
3-M syndrome; patient had presumed syndromic growth disorder

but multiple genetic, radiographic, and clinical evaluations did not
lead to diagnosis
(1/1)
Exome sequencing of patient, parents and unaffected brother

identified known but rare recessive growth disorder with normal
intelligence; patient can be counseled regarding reproduction;
referred to urology regarding fertility preservation due to
hypogonadism
2013
Study
Diagnostic
Yield (%)
Results

Johnson et al.
2012
BrownVialettoVan Laere syndrome; Patient had similar

syndrome but later age of onset and slower progression
(1/1)
Family homozygosity mapping and patient exome sequencing

identified 2 potential variants, one confirmed by follow-up
Sanger sequencing in additional similar patients; another patient
with same variant started on riboflavin therapy due to variant
identification and biological ramifications, with improvement in
pulmonary function and upper limb strength
Samuels et al.
2013
Glucocorticoid deficiency; 2 unrelated patients presented at young

ages with severe symptoms of hypoglycemia, normal electrolytes,
low cortisol, and high ACTH.
(2/2)
Exome sequencing in one patient identified 2 heterozygous

mutations in POMC, a precursor protein from which ACTH is
cleaved; Sanger sequencing for POMC in the second patient
identified one of the same mutations in homozygous form;
functional and immunoreactive studies showed an excess of
inactive ACTH in both patients; as a result, mineralocorticoid
treatment was discontinued
Pierson et al.
2012
GM1-gangliosidosis; this diagnosis originally considered for

the severely affected 19-year old patient but an early betagalactosidase enzyme analysis was reported normal and the
search for a diagnosis continued for years
(1/1)
Exome sequencing of the patient and 4 family members identified

2 GLB1 variants c/w the diagnosis; repeat of beta-galactosidase
enzyme was c/w diagnosis and additional labs supported;
average time to diagnosis for this condition may be >5 years;
exome sequencing evaluates without prior bias
Puffenberger
et al. 2012
Various diagnoses; rural pediatric practice serving Amish and

Mennonite children
(2/7 firm,
5/7 likely)
Exome sequencing was used to search for diagnoses in children

with uninformative SNP mapping or potentially large gene lists
Berger et al.
2011
Prenatal ventriculomegaly; family of 3 with the condition
(1/1)
Linkage analysis plus exome sequencing in one sibling resulted

in the economical identification of a mutation in the AIFM1 gene
that segregated with disease and was absent in 86 unrelated
controls
Solomon et al.
2011
Neonate with cardiac malformations suffered from unexplained

post-surgical severe pulmonary artery hypertension
(1/1)
CPSI mutation in combination with environmental trigger

(surgery) may have resulted in pulmonary artery hypertension
due to inadequate nitric oxide production; identification of the
genetic risk allows for medical prevention for the patient and
relatives with the same mutation
22
Study
Study
Diagnostic
Yield (%)
Results

Bonnefond
et al. 2010
Neonatal diabetes mellitus (NDM); neonate presented with

NDM but was negative for mutations in known genes
(1/1)
Exome sequencing identified a mutation in a known gene

that was originally missed by, but then confirmed by Sanger
sequencing; confirmation of diagnosis is necessary to guide
selection of oral sulfonylurea drugs instead of insulin therapy
Rios et al.
2010
Sitosterolemia; infant with xanthomas and very high plasma

cholesterol but no family history
(1/1)
Exome sequencing revealed 2 nonsense mutations in ABCG5,

resulting in a diagnosis with an atypical presentation; confirmed
after weaning
Studies assessing exome sequencing methodology

Dias et al.
2012
125
Muscle disease and spastic paraplegia
Assessed targeting efficiency and sequencing coverage of 88

genes associated with disease; >9599% of targeted known
mutation positions were sequenced to at least 1x coverage and
5587% to at least 20x coverage in every targeted gene exon,
suggesting reliable detection of mutations; regions of poor
coverage may need follow-up Sanger sequencing
Berg et al.
2011
Primary ciliary dyskinesia (PCD); patients were enrolled in

a study of clinical and molecular aspects of PCD
(4/6 known
mutations
detected)
Pilot study to investigate the performance characteristics of

exome sequencing in detecting previously identified mutations as
well as benign variants; compared to Sanger sequencing, exome
sequencing had a sensitivity of 98.3%; discordant results were
small insertions/deletions, which make up 22% of known human
gene mutations
Licastro et al.
2012
12
Usher syndrome; clinically diagnosed patients who were

negative for previously described mutations in known
Usher-associated genes
83
Exome sequencing and targeted next-gen sequencing compared

for diagnosis; targeted sequencing was more efficient but less
effective given high genetic heterogeneity; exome sequencing is
limited by the need for high coverage and accurate interpretation
of novel variants
targeted testing on other individuals were also

used to complete the diagnostic assessment.
There were 6 larger patient series, sequencing 12 to 118 exomes per study. An important, although intermediate, outcome of the
larger patient series is diagnostic yield, i.e.,
the proportion of patients with a diseasecausing variant identified by exome sequencing. Normally the diagnostic yield of exome
sequencing would be compared with that
obtained using current, standard Sanger
sequencing in similar populations. In these
studies, patients have undergone prior testing
and remain undiagnosed, thus the diagnostic
yield in these studies represents additional
yield beyond that of conventional testing.
Because of different patient selection criteria, depending on disease area or degree of
prior evaluation, diagnostic yield in the larger
patient series varied considerably from 10% to
54%. The lowest diagnostic yield came from
the National Institutes of Health Undiagnosed
Diseases Program that includes highly selected,
difficult to diagnose patients with extensive
prior testing.5 Results were more often fully
successful in the 21 small patient/family studies
(sequencing 110 exomes per study). Since
these studies are largely positive or negative on
the basis of the index case, and few if any negative results were found in this group of studies,
publication bias could be a factor.
The outcomes of greatest interest are those
affecting medical management of the index
patient, reproductive decisions (e.g., of the
patient, the parents regarding subsequent
pregnancies, or of family members identified as
carriers), and possibly identification of family
members who may be affected by the same disorder. However, there were few reports of these
kinds of outcomes as a result of molecular diagnosis by exome sequencing. Any related information provided in the reports is summarized
and highlighted in bold in Table 5, showing
anecdotal positive clinical consequences of the
diagnosis. In a few cases, medical management of patients was changed. Importantly,
exome sequencing revised the diagnosis for
some patients. However, no systematic study of
outcomes related to medical management or
reproductive decisionmaking was identified.
Studies comparing exome sequencing to more
traditional genetic testing technology generally
5
agree that exome sequencing is more efficient

and more likely to result in a diagnosis. Berg
et al. report a sensitivity of 98.3% for detecting previously identified mutations as well as
benign variants (Berg et al. 2011). Particular
limitations noted were gaps in gene and exon
coverage and difficulty interpreting variants.
For example, Dias et al. specifically focused on
88 genes known to be associated with muscle
disease and spastic paraplegia (Dias et al.
2012). Although the exome capture kits tested
targeted the NCBI Consensus CDS (from coding
DNA sequence) annotated genes, only 82 of the
88 genes are included in this database (http://
www.ncbi.nlm.nih.gov/CCDS). In addition,
some well-annotated genes had poor exon
coverage and not all nucleotides of interest
were captured. The authors noted that during
the time of their study, exome capture kits
had already expanded gene enrichment, but
that would not address poor coverage within
targeted regions, which is a function of current
sequencing technology.
Berg et al. cautioned that clinical applications of
massively parallel sequencing technology need
to be validated on different types of reference
mutations to determine performance characteristics; such validation should include the analytic software used to filter variants (Berg et al.
2011). In particular, Berg et al. reported a discordance between traditional Sanger sequencing and next-generation sequencing for small
insertion or deletion variants, which account for
about 22% of the approximately 134,000 total
mutation entries in the human gene mutation
database (http://www.hgmd.cf.ac.uk/). Licastro
et al. who focused on genes related to Usher
syndrome, noted that despite using high stringency multi-step filter criteria to select
the pathogenic variants, they found a high
number of novel, potentially pathogenic variants requiring considerable interpretation
(Licastro et al. 2012).
Discussion
Depending on the disorder and the degree of
genetic and clinical heterogeneity, the current
diagnostic pathway for patients with suspected
genetic disorders accompanied by multiple
anomalies may depend on various combinations
of low-yield radiographic, electrophysiological, biochemical, biopsy, and targeted genetic
The entire diagnostic experience of the program, including all patients seen, is reviewed here: http://www.genome.gov/27543155
24
evaluations (Dixon-Salazar et al. 2012). The

search for a diagnosis may become a time-consuming and expensive odyssey that is frustrating for patients, families and providers. Exome
sequencing using next-generation sequencing
technology is a relatively new and unbiased
approach to obtaining a genetic diagnosis in
patients with potentially less testing compared
with traditional methods. Published exome
sequencing studies show that the technology
can be used to detect previously annotated
pathogenic mutations and reveal new likely
pathogenic mutations in known and unknown
genes. The diagnostic yield, based on a limited
number of studies, appears to be significantly
increased above that of traditional Sanger
sequencing.
However, exome sequencing does not provide
a diagnosis for every patient tested and is not
without limitations. Gaps exist in the captured
exome that may not be corrected by increasing
depth of sequencing; it is difficult to filter and
interpret potential causative variants from the
large number of variants of unknown significance generated for each patient; existing databases that catalog variants and putative disease
associations are known to have significant
entry error rates (Bell et al. 2011; Dixon-Salazar
et al. 2012; McDonald et al. 2012). The technology itself has not yet been well-standardized for
the clinical laboratory, has not been fully characterized in publicly available documents with
regard to the analytic validity for the various
types of relevant mutations, and the few existing professional guidelines give only high-level
direction. Exome sequencing has similar limitations as Sanger sequencing; e.g., it will not
identify: intronic sequences or gene regulatory
regions; chromosomal changes; large deletions,
duplications or rearrangements within genes;
nucleotide repeats; or epigenetic changes.
Finally, there are ethical questions about
reporting incidental findings, such as medically
relevant mutations in genes unrelated to the
diagnostic question, mutations of unknown significance, sex chromosome abnormalities
or non-paternity.
The clinical utility of exome sequencing (and
any other diagnostic test) lies in the influence
of the results on medical decision-making
and patient outcomes. Currently there are no
published studies that systematically examine

potential outcomes of interest such as changes
in medical management (including revision of
initial diagnoses), and changes in reproductive
decisionmaking. A small number of studies
of patient series, and a larger number of very
small series or family studies report anecdotal
examples of medical management and reproductive decision-making outcomes of exome
sequencing in patients who were not diagnosed
by traditional methods. These studies show that
over and above traditional molecular and conventional diagnostic testing, exome sequencing
can lead to a diagnosis that influences patient
care and/or reproductive decisions, but give
no indication of the proportion of patients for
which this is true. The publication of a large
number of small diagnostic studies with positive results but few with negative results, raise
the possibility of publication biasthe impact
of which is unknown.
Conclusions
Exome sequencing using next-generation
sequencing has been recently introduced as
a laboratory-developed diagnostic clinical
laboratory test. A major indication for use is
molecular diagnosis of patients with suspected
genetic disorders or of patients with known
genetic disorders with substantial genetic
heterogeneity involving substantial gene complexity. Such patients may be left without a
satisfactory clinical diagnosis of their disorder
despite a lengthy diagnostic odyssey involving
a variety of traditional molecular and other
types of conventional diagnostic tests. For some
patients, ordering exome sequencing soon
after initial conventional testing has failed may
avoid the diagnostic odyssey and return a likely
pathogenic variant. Currently, the diagnostic
yield for single-gene disorders appears to be
no greater than 50% and possibly less, depending on the patient population and provider
expertise. Medical management options may
be available for only a subset of those diagnosed. Reproductive decisions by parents of an
affected child or affected patients of reproductive age, however, will be informed by understanding the mode of inheritance. Appropriate
use of exome sequencing requires considerable
genetic, clinical, and genetic counseling expertise.
NOTICE OF PURPOSE: TEC Assessments are scientific opinions, provided solely for informational purposes. TEC Assessments
should not be construed to suggest that the Blue Cross Blue Shield Association, Kaiser Permanente Medical Care Program or the
TEC Program recommends, advocates, requires, encourages, or discourages any particular treatment, procedure, or service; any
particular course of treatment, procedure, or service; or the payment or non-payment of the technology or technologies evaluated.
CONFIDENTIAL: This document contains proprietary information that is intended solely for Blue Cross and Blue Shield Plans
and other subscribers to the TEC Program. The contents of this document are not to be provided in any manner to any other
parties without the express written consent of the Blue Cross and Blue Shield Association.
26
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Technology Evaluation Center

Special Report: Exome

Technology Evaluation Center

Technology Evaluation Center

Published in cooperation with Kaiser Foundation Health Plan and

Single-gene disorders may be expressed in

Technology Evaluation Center

for family members and provide helpful

frequently is limited in scope and requires

Inherited disorders are autosomal if they

Inheritance patterns can sometimes be determined by analyzing the family history or

fewer than 5 disorders had known molecular

Confirm a suspected or clinically

In contrast, a large proportion of single-gene

As an example, Miller et al. (2011) note that

Molecular Diagnosis. Given the complexity

Several guidelines address molecular diagnosis,

Technology Evaluation Center

or syndromes). Consistent themes are that

genome sequencing is currently limited in

Table 1. Examples of Guidelines/Policies that Address Molecular Testing in Hereditary Disorders

Impact of Genetic Testing

Exome sequencing should be considered for clinical diagnosis

Policy specifically references exome

Differentiate between cerebral palsy and other neurometabolic

Evaluation of the child

Microcephaly has been associated with numerous genetic

Targeted testing may be considered

Hereditary ataxias must be distinguished from non-hereditary

Targeted testing for specific syndromes,

Channelopathies: molecular testing may aid diagnosis but

Targeted testing for specific syndromes,

Type of Genetic Testing/

Impact of Genetic Testing

Type of Genetic Testing/

Diagnosis and treatment

Diagnosis is clinical; genetic testing can aid in classification,

Targeted testing for some specific forms

Diagnostic workup follows a stepwise procedure:

Various types of genetic tests depending

Mutation testing in the DMD gene is necessary to confirm

Deletion/duplication analysis followed by

Detection of an associated mutation is the standard of

Targeted testing for specific syndromes,

Genetic testing should be conducted for the accurate diagnosis

Targeted testing per decision algorithm

Technology Evaluation Center

In 2009, Ng et al. (2009) published a proof of

individuals from the same family. For example,

See CAP press release available at http://www.cap.org/apps/cap.portal?_nfpb=true&cntvwrPtlt_actionOverride=%2Fpor

Gold standard for detecting unknown point

Maximum feasible size of clinical sequencing unit

Single-gene exon(s), single gene (if not excessively large)

Exome (whole genome likely, in future)

Ability to identify a previously established causal

Relatively efficient; if a small number of known mutations

Inefficient for most circumstances of a known causal variant;

Requires accurate and complete differential diagnosis

The exome or a panel of genes known to be associated with the

variant in a single gene

Ability to identify a causal variant for a suspected

Unknown genes will be missed.

Technology Evaluation Center