Next‐generation sequencing for the genetic screening of phaeochromcytomas and paragangliomas: riding the new wave, but with caution

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. More than one-third of all PHEO/PGLs have a pathogenic germline mutation 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. 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 nucleotides 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. 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 technologies based on massive parallel sequencing, also referred to as next-generation sequencing (NGS), have revolutionized the detection of disease-associated genes. In particular, wholeexome sequencing (WES), in which the entire coding portion of the genome is targeted and where most disease-associated mutations 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. With the steady decline in costs and concomitant expansion of its user base, NGS has now expanded beyond its role as a discovery tool to gradually replace conventional methods for diagnostics in many genetic disorders, including inherited cancers. In this capacity, NGS methods have also been adapted to target only specific candidate genes or loci, instead of the entire exome. 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. 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. report their use of WES for the diagnosis of germline mutations in PHEO/PGLs genes. In this study, two different exome 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 experiments and found it to be inferior to the expected by the manufacturers, although the relatively low depth of sequence of the study samples may have accounted for their suboptimal performance. In summary, McInerney-Leo et al. have shown that WES is a cost-effective method, capable of identifying pathogenic mutations 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.