Test ID: SDHKM
Succinate Dehydrogenase (SDH) Gene, Known Mutation

Diagnosis of suspected succinate dehydrogenase (SDH) disease, when familial mutations have been previously identified

Screening presymptomatic members of SDH families, when familial mutations have been previously identified

Tailoring optimal tumor-surveillance strategies for patients, when used in conjunction with phenotyping, when familial mutations have been previously identified

• Informed Consent for Genetic Testing
• SDHB, SDHC, SDHD Gene Testing Patient Information Sheet

Polymerase Chain Reaction (PCR) Amplification/DNA Sequencing and Deletion Detection by Multiplex Ligation-Dependent Probe Amplification (MLPA)

(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)

Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

Collection Instructions: Send specimen in original tube.

Additional Information: This test is only applicable if a mutation has previously been identified in a family member of this individual.

Forms:

1. SDHB, SDHC, SDHD Gene Testing Patient Information Sheet (Supply T659) in Special Instructions.

2. New York Clients-Informed consent is required. Please document on the request form or electronic order that a copy is on file. An Informed Consent for Genetic Testing (Supply T576) is available in Special Instructions.

Succinate dehydrogenase (SDH) is a mitochondrial membrane-bound enzyme complex consisting of 4 subunits: SDHA, SDHB, SDHC, and SDHD. SDH is an oxidoreductase that catalyzes the oxidation of succinate to fumarate (tricarboxylic acid cycle function) and the reduction of ubiquinone to ubiquinol (respiratory chain function).

Homozygous loss of function mutations or homozygous deletions of SDH subunit genes are embryonal lethal, with the exception of some biallelic SDHA mutations, which cause Leigh syndrome. No disease-associated heterozygote SDHA mutations or deletions have been reported. By contrast, heterozygous mutations and deletions of SDHB, SDHC, or SDHD result in a high life-time penetrance autosomal dominant tumor syndrome. Patients have only 1 functioning germline copy of the affected SDH subunit gene. When the second, intact copy is somatically lost or mutated in target tissues, tumors develop. Sympathetic and parasympathetic ganglia are preferentially affected, resulting in development of paragangliomas (PGLs) or pheochromocytomas (PCs). PGLs might include parasympathetic ganglia (neck and skull-base) or sympathetic ganglia (paravertebral sympathetic chain from neck to pelvis). PCs can involve 1 or both adrenal glands. Almost all PCs overproduce catecholamines, resulting in hypertension with a predilection for hypertensive crises. About 20% of PGL, mostly intra-abdominal, also secrete catecholamines. PGLs in the neck do not usually produce catecholamines. SDH-associated PGLs and PCs are typically benign; however, malignancy has been described in a minority of patients (especially in patients with SDHB mutations). In addition, because of the germline presence of the mutation or deletion, new primary tumors might occur over time in the various target tissues. Finally, tumors unrelated to chromaffin tissues, namely renal cell carcinoma (RCC: SDHB only) and gastrointestinal stromal tumors (GISTs: SDHB, SDHC, and SDHD), affect a minority of patients.

Collectively, heterozygous germline mutations and deletions of SDHB, SDHC, or SDHD are found in 30% to 50% of apparently sporadic PGL cases and can be confirmed in >90% of clinically hereditary cases. The corresponding figures are 1% to 10% and 20% to 30% for outwardly sporadic PC and seemingly inherited PC, respectively. The prevalence of SDHD mutations and deletions is higher than that of SDHB, which in turn exceeds the figures for SDHC. SDHB and SDHC mutations show classical autosomal dominant inheritance, while SDHD mutations show a modified autosomal dominant inheritance with chiefly paternal transmission, suggesting maternal imprinting (the molecular correlate of which remains unknown). SDHB is most strongly associated with PGL (usually functioning), but adrenal PCs also occur, as do occasional GISTs and RCCs, with the latter being found exclusively in this subtype. SDHD shows a disease spectrum similar to SDHB, except head and neck PGLs are more frequent than in SDHB, while functioning or malignant PGLs/PCs and GISTs are less common. SDHC has thus far been mainly associated with PGLs of skull base and neck. Abdominal/functioning PGLs or PCs are uncommonly seen in patients with SDHC mutations, and GISTs are very rare. However, there is limited certainty about the SDHC genotype-phenotype correlations, as the reported case numbers are low.

Genetic testing for SDHB, SDHC, and SDHD germline mutations and deletions is highly accurate in identifying affected patients and presymptomatic individuals. It is advocated in all patients that present with PGL. Accurate diagnosis assists in designing optimal follow-up strategies, since the rate of new or recurrent tumors is much higher in patients with SDH mutations or deletions than in true sporadic cases.

Screening for mutations in SDH genes is not currently advocated for sporadic adrenal PC, but is gaining in popularity, often alongside tests for mutations of other predisposing genes: RET (multiple endocrine neoplasia type 2, MEN2), VHL (von Hippel-Lindau syndrome), and NF1 (neurofibromatosis type 1). Seemingly familial PC cases, who do not have an established diagnosis of a defined familial tumor syndrome, should be screened for SDH gene mutations, along with screening of the other predisposing genes listed above.

In order to minimize the cost of genetic testing, the clinical pattern of lesions in PGL and PC patients might be used to determine the order in which the 3 disease-associated SDH genes are tested. Genetic diagnosis of index cases allows targeted presymptomatic testing of relatives.

An interpretive report will be provided.

The presence of a known disease-causing mutation or deletion confirms the diagnosis of succinate dehydrogenase (SDH)-associated paraganglioma (PGL) or pheochromocytoma (PC).

The presence of DNA sequence variations or deletions that have not been previously linked to PGL or PC is classified as a sequence variation of unknown significance. Within this group, the observed variants are classified as:

-Likely to be disease-causing: this includes all DNA sequence variations that are predicted to result in a nonfunctioning protein. Examples include premature stop-codons, small indels causing frameshifts, mutated start codons, changes that definitively alter splice sites, and large deletions affecting > or =1 exon.

-Possibly disease-causing: this includes all DNA sequence variations for which it is uncertain whether they will or will not result in a protein with impaired function. Examples include previously unreported missense mutations, in-frame exonic small indels, and intronic mutations or small indels that have some potential of affecting splicing. For this category of sequence changes we will usually provide some additional guidance in an expanded interpretive report.

-Unlikely to be disease-causing: this includes all sequence variations of the gene that will probably not result in any changes to the translated protein. Examples include silent mutations (no amino acid change), intronic variants that are not predicted to affect splicing, sequence changes that are seen in conjunction with another, known disease-causing changes (SDH mutations are autosomal dominant), and sequence changes that have been seen many times during routine testing in both affected and unaffected patients.

Many mutations have been correlated with certain phenotypes, as indicated in the "Clinical Information" section. When such correlations are known for a detected mutation or deletion, they are included in the report.

Tumor Testing:

The detection of a disease-causing mutation, deletion, insertion, or a large deletion, indicates that SDH gene inactivation has occurred. In a tumor, such inactivation is usually biallelic.

Rarely, unknown polymorphisms in primer- or probe-binding sites can result in false-negative test results (DNA sequencing) or either false-positive or false-negative results (multiplex ligation-dependent probe amplification [MLPA] deletion screening), due to selective allelic drop-out. False-negative or false-positive results can occur in MLPA deletion screening assays due to poor DNA quality.

The current test does not examine the promoters, other gene regulatory elements, or most of the intronic portions of the SDHB, SDHC, and SDHD genes. The impact of this on detection rates is unknown. Based on observations in other genetic disorders, it is generally believed that <5% of disease-causing mutations occur in these regions.

There may be (several) other, as yet unidentified, genes that can cause a phenotypically similar picture as succinate dehydrogenase (SDH) mutations and deletions.

Collectively, the above causes, along with various other preanalytical and analytical problems that are not unique to genetic testing (eg, specimen mix-up), probably account for the estimated false-negative rate of <10% (likely <5%) that is observed with genetic SDH testing.

The absence of any sequence variations and deletions within SDHB, SDHC, and SDHD, as compared to the wild-type reference sequence, or the presence of only known normal variant sequence polymorphisms, excludes SDH-associated paraganglioma (PGL) or pheochromocytoma (PC) with at least 90% certainty.

When testing for known mutations in family members of index cases, the presence or absence of the known mutation predicts with near 100% certainty, whether the tested person has inherited SDH-associated PGL or PC.

If the specimen is from a tumor (frozen tissue), in particular a sporadic tumor (rather than a SDH-related tumor), 1 of the alleles might be inactivated by promoter hypermethylation. Our assay does not detect hypermethylation.

This test does not reliably detect deletions in formalin-fixed, paraffin-embedded tissues.

We sequenced the SDHB, SDHC, and SDHD genes in 42 specimens that had previously been tested for succinate dehydrogenase (SDH) mutations at the National Institutes of Health (NIH). We were blinded to the original results until completion of all sequencing. All previously found mutations were confirmed. Overall, 27 patients had SDHB mutations, 2 patients had SDHC mutations, and 8 patients had SDHD mutations. Inter- and intra-assay testing showed 100% concordance for all sequenced regions. Fifteen specimens from healthy individuals were also sequenced. All showed wild-type sequence for SDHB, SDHC, and SDHD.

Another 42 specimens from the NIH were tested for deletions of SDHB, SDHC, and SDHD, using multiplex ligation-dependent probe amplification-Luminex Flexmap technology. Seventeen specimens were found to have deleted portions of 1 of the SDH genes. These results were confirmed by the NIH. In addition, 50 specimens from healthy individuals were tested for deletions. We detected no deletions of SDHB, SDHC, or SDHD in any of these individuals.

1. Briere JJP, Favier J, Gimenez-Roqueplo AP, Rustin P: Tricarboxylic acid cycle dysfunction as a cause of human diseases and tumor formation. Am J Physiol Cell Physiol 2006 Dec;291(6):C1114-1120

2. Young WF Jr: Paragangliomas: Clinical Overview. Ann NY Acad Sci 2006 Aug;1073:21-29

3. Bornstein SR, Gimenez-Roqueplo AP: Genetic Testing in Pheochromocytoma: Increasing Importance for Clinical Decision Making. Ann NY Acad Sci 2006;1073:94-103

4. Benn DE, Richardson AL, Marsh DJ, Robinson BG: Genetic Testing in Pheochromocytoma and Paraganglioma-Associated Syndromes. Ann NY Acad Sci 2006;1073:104-111

SDHB, SDHC, and SDHD gene sequences are generated by PCR amplification of all 18 exons, including flanking intronic sequence, followed by automated fluorescent-dye terminator DNA sequencing. Detection of large deletions involving SDHB, SDHC, and SDHD, or parts of these genes, is accomplished by multiplex ligation-dependent probe amplification and detection on Luminex beads specific to each exon. (Unpublished Mayo method)

Monday through Friday; Varies

81403-Known familial variant, not otherwise specified, for gene listed in Tier 1 or Tier 2, DNA sequence analysis