C O M M E N T A R Y

Next-generation sequencing for the genetic screening ofphaeochromcytomas and paragangliomas: riding the new wave,but with caution*

Rodrigo A. Toledo* and Patricia L. M. Dahia*,†

*Division of Hematology and Medical Oncology, Department of Medicine, Cancer Therapy and Research Center and †GreeheyChildhood Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

(Received 23 October 2013; accepted 24 October 2013)

Phaeochromcytomas (PHEO) and paragangliomas (PGL) are

catecholamine-secreting tumours derived from chromaffin cells

of the adrenal medulla or sympathetic paraganglia, respectively,

which carry high genetic and allelic heterogeneity.1 More than

one-third of all PHEO/PGLs have a pathogenic germline muta-

tion in one of several susceptibility genes, and genetic testing is

now recommended for all patients. Furthermore, somatic driver

mutations were recently recognized as frequent events in these

tumours.2–5 In all, 16 different PCC/PGL-related genes have

been identified (VHL, RET, NF1, SDHA, SDHB, SDHC, SDHD,

SDHAF2, MAX, TMEM127, HIF2A/EPAS1, KIF1B, PDH2, FH

and HRAS), involving a total of 20 791–30 078 coding nucleo-

tides spanned by 141–217 exons, depending on the gene isoform

analysed. This has become a laborious and costly process for

genetic laboratories. To optimize the screening of patients with

PHEO/PGLs, algorithms that prioritize genetic testing have been

developed.6,7 Although proven to decrease costs and reduce

analysis turnaround time, these stepwise approaches are more

effective for patients presenting with syndromic features or a

positive family history.

In recent years, high-throughput DNA-sequencing technolo-

gies based on massive parallel sequencing, also referred to as

next-generation sequencing (NGS), have revolutionized the

detection of disease-associated genes.8 In particular, whole-

exome sequencing (WES), in which the entire coding portion of

the genome is targeted and where most disease-associated muta-

tions lie, has become the NGS ‘workhorse’. In WES, DNA is

fragmented, captured by hybridization to platforms spanning the

exome, ligated to adaptors and massively parallel-sequenced.

Notably, four of the PHEO/PGL-related genes were discovered

through WES.4,5,9,10 With the steady decline in costs and con-

comitant expansion of its user base, NGS has now expanded

beyond its role as a discovery tool to gradually replace conven-

tional methods for diagnostics in many genetic disorders,11

including inherited cancers.12 In this capacity, NGS methods

have also been adapted to target only specific candidate genes or

loci, instead of the entire exome.12

The genetically heterogeneous nature of PHEO/PGLs makes

them ideally suited for NGS-based diagnostic screens. A recent

study targeting nine PCC/PGL susceptibility genes by polymerase

chain reaction (PCR) followed by massively parallel sequencing

identified 98�7% of known mutations in a cohort of 85 patients,

demonstrating feasibility of the strategy.13 However, as the

screening was only limited to the optimized candidate genes, a

mutation was found in less than 17% of a discovery cohort,

which speaks of the need for expanded diagnostic platforms in

PHEO/PGLs.

In the current issue of Clinical Endocrinology, McInerney-Leo

et al.14 report their use of WES for the diagnosis of germline

mutations in PHEO/PGLs genes. In this study, two different ex-

ome capture platforms were used in a group of 11 previously

characterized germline samples, one of which was used in both

platforms. Applying basic bioinformatic analysis, the expected

mutation was found in all but one case, at a faster rate and

lower cost than conventional sequencing. One mutation, on the

SDHC gene, was missed by one capture method, but it was

detected by the second platform. This prompted McInerney-Leo

et al. to systematically examine the expected capture efficiency,

defined as targeting of >90% of the coding sequence, of 12

PHEO/PGL genes in these two platforms, as well as in three

other off-the-shelf methods, by examining data files downloaded

from the manufacturers’ website. They found great variability on

the coverage of the 12 genes across the five platforms, including

poor coverage of the SDHC gene. Strikingly, no single platform

showed complete coverage of all the PHEO/PGLs exons. They

also assessed the actual capture achieved by their own experi-

ments and found it to be inferior to the expected by the manu-

facturers, although the relatively low depth of sequence of the

study samples may have accounted for their suboptimal perfor-

mance.

In summary, McInerney-Leo et al. have shown that WES is a

cost-effective method, capable of identifying pathogenic muta-

tions in PHEO/PGL susceptibility genes while substantially

reducing bench work and turnaround time of the screening

process. However, the study underscores a limitation of current

*Please see related paper on pages 25–33 of this issue.

Correspondence: Patricia L. M. Dahia, Division of Hematology andMedical Oncology, Department Medicine, Cancer Therapy and ResearchCenter, University of Texas Health Science Center at San Antonio, SanAntonio, TX, USA. Tel.: +1 210 567 4866; E-mail: dahia@uthscsa.edu

© 2013 John Wiley & Sons Ltd 23

Clinical Endocrinology (2014) 80, 23–24 doi: 10.1111/cen.12357

platforms and the need to occasionally complement non- or

under-represented target areas with independent techniques to

avoid missing a mutation. Additional technical limitations, not

discussed in this study, may result from the presence of pseud-

ogenes or repetitive regions that may lead to mapping and align-

ment errors. These shortcomings may become even more

relevant when tumour, instead of germline, DNA (as in the

present study) is used for sequencing, due to tumour heteroge-

neity and the need for more sophisticated bioinformatic analy-

sis.4,15 NGS-based diagnostics is still an evolving area, and the

quality and efficiency of capture methods are expected to

improve over time. Moreover, defining the ideal NGS platform

for screening may require extending the target region beyond

the exome. Outside the scope of this commentary, but no less

important, will be devising strategies to address variants of

unknown significance in PHEO/PGL genes and ‘incidental’

genetic findings (i.e. PHEO/PGL-unrelated, but potentially med-

ically actionable mutations) intrinsically associated with gen-

ome-wide sequencing approaches, before NGS enters the

mainstream of diagnostic testing in these tumours.

The transition of Sanger sequencing to NGS in the molecular

diagnostics of diseases with high genetic and allelic heterogeneity

appears inevitable11; however, adoption of the new methodology

must happen with caution to prevent errors and misinterpreta-

tions that can carry negative clinical impact.

Acknowledgements

P.L.M.D is a recipient of a Voelcker Investigator Award, a

Cancer Prevention Institute of Texas (CPRIT) Individual Investi-

gator Award, a Department of Defense Peer Reviewed Medical

Research Program Investigator-Initiated Research Award (DOD-

PRMRP-PR110571) and is supported by funds from the Greehey

Children Cancer Research Institute, UTHSCSA.

Disclosure

The authors have nothing to disclose.

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© 2013 John Wiley & Sons Ltd

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24 R. A. Toledo and P. L. M. Dahia