Test ID: SDHSP
Succinate Dehydrogenase (SDH) Gene Analysis

Diagnosis of familial SDHB, SDHC, or SDHD gene mutations or deletions in patients with paragangliomas or pheochromocytomas

Used in conjunction with phenotyping to tailor optimal tumor-surveillance strategies

If SDH gene sequencing does not identify a mutation, then SDH deletion detection will be performed at an additional charge.

• 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: Transfusions will interfere with testing for up to 4 to 6 weeks. DNA obtained from white cells may not provide useful information for patients who received a recent transfusion of blood that was not leukocyte-reduced. Wait 4 to 6 weeks until transfused cells have left the patient's circulation before drawing the patient's blood specimen for genotype testing.

Forms:

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

2. Informed Consent for Genetic Testing (Supply T576) in Special Instructions is required.

3. 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 usually do not 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 numbers 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 RCCs 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 and 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 and recurrent tumors is much higher in patients with SDH mutations or deletions than in truly 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 previously listed.

In order to minimize the cost of genetic testing, the clinical pattern of lesions in PGL and PC patients may 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.

All detected alterations will be evaluated according to American College of Medical Genetics and Genomes (ACMG) recommendations.(1) Variants will be classified based on known, predicted, or possible pathogenicity and reported with interpretive comments detailing their potential or known significance.

Rare unknown polymorphisms in primer- or probe-binding sites can rarely result in allelic drop out and false-negative genetic tests.

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. It is unknown what the impact of this is on detection rates. Based on observations in other genetic disorders, it is generally believed that <5% of disease-causing mutations occur in these regions.

In addition to disease-related probes, the multiplex ligation-dependent probe amplification technique utilizes probes localized to other chromosomal regions as internal controls. In certain circumstances, these control probes may detect other diseases or conditions for which this test was not specifically intended. Results of the control probes are not normally reported. However, in cases where clinically relevant information is identified, the ordering physician will be informed of the result and provided with recommendations for any appropriate follow-up testing.

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 preanalytic and analytic 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.

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 mutations previously identified 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. Richards CS, Bale S, Bellissimo DB, et al: ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med 2008 Apr;10(4):294-300

2. Briere JJ, 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

3. Young WF Jr: Paragangliomas: clinical overview. Ann NY Acad Sci 2006;1073:21-29

4. Bornstein SR, Gimenez-Roqueplo AP: Genetic testing in pheochromocytoma: increasing importance for clinical decision making. Ann NY Acad Sci 2006;1073:94-103

5. 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.(GenBank accession numbers: NM_003000.2, NM_003001.3, NM_003002.2 respectively) 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 8 a.m.

81404-SDHD (succinate dehydrogenase complex, subunit D, integral membrane protein) (eg, hereditary paraganglioma), full gene sequence                                                                  

81405-SDHC (succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa) (eg, hereditary parganglioma-pheochromocytoma syndrome), full gene sequence            

81405-SDHB (succinate dehydrogenase complex, subunit B, iron sulfar) (eg, hereditary paraganglioma), full gene sequence