Table 5 Supplementary and/or functional tests available for PPGL genes

From: Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas

Type of material required

Test description

Expected results

Target gene

Target mutation type

Evidence level of test*

Refs

Tumour or leukocyte RNA

cDNA sequencing

Identification of aberrant RNA transcript

All PPGLs susceptibility genes

Splice mutations

B

108,114

Tumour DNA

Sequencing, MLPA, SNP array or microsatellite markers study (for copy number analysis)

Identification of LOH (by deletion or somatic mutation of the contralateral allele)

PPGLs tumour suppressor genes

All

B

105,107, 110,111

Methylation analysis

Identification of methylation of the contralateral allele

PPGLs tumour suppressor genes

All

B

21

Frozen tumour tissue

SDH enzymatic activity measurement

Loss of SDH enzymatic activity

SDH

All

A

115

Fumarase enzymatic activity measurement

Loss of fumarase enzymatic activity

FH

All

A

20

Succinate concentration

Accumulation of succinate

SDH

All

B

20

Fumarate concentration

Accumulation of fumarate

FH

All

B

20

Metabolome profiling by HRMAS NMR spectroscopy

Accumulation of succinate

SDH

All

C

124

FFPE tumour tissue section

SDHB/SDHD IHC

SDHB negative or SDHD positive IHC

SDH

All

A

5,116,117,118

SDHA IHC

SDHA negative IHC

SDHA

All

A

112,117,119

MAX IHC

MAX negative IHC

MAX

Truncating mutations

B

22

2-SC IHC

2-SC positive IHC

FH

All

B

101

In vitro assay

PC12 rat phaeochromocytoma cell-based luciferase reporter assay (mutagenesis)

Alteration of MAX regulatory effects on MYC

MAX

Missense

C

120

Endomembrane localization or subcellular distribution of expressed mutant constructs by confocal microscopy

Intracellular distribution of TMEM127 mutant

TMEM127

Missense

C

94,114,121

SDH cellular distribution

Loss of SDH mitochondrial localization

SDH

All

C

122

In vivo assay

In vivo succinate detection by magnetic resonance spectroscopy

Accumulation of succinate in vivo

SDH

All

C

123

  1. FFPE, formalin-fixed and paraffin-embedded; IHC, immunohistochemistry; LOH, loss of heterozygosity; MLPA, multiplex ligation-dependent probe amplification; PPGLs, phaeochromocytomas and/or paragangliomas; RT-PCR, reverse-transcriptase PCR; SDH, succinate dehydrogenase; 2-SC, succinyl-cysteine, SNP, single nucleotide polymorphism.
  2. *Evidence level of test: A, widely used, with strong functional accuracy, and independently validated by multiple laboratories; B, validated by more than one laboratory, moderate functional accuracy; C, initial testing only; functional accuracy unknown.
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